Detergent compositions comprising gh9 endoglucanase variants ii

ABSTRACT

Detergent compositions may include endoglucanase variants and methods of using such detergent compositions. The endoglucanase may have an alteration at one or more positions where the variant has at least 60% but less than 100% sequence identity to SEQ ID NO: 2.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a national stage entry according to 35 U.S.C.§ 371 of PCT application No.: PCT/EP2018/0171105 filed on Aug. 3, 2018;which claims priority to German Patent Application Serial No.: 10 2017214 809,8, which was filed on Aug. 24, 2017; which is incorporatedherein by reference in its entirety and for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

TECHNICAL FIELD

Detergent compositions comprising novel GH9 endoglucanase variants mayexhibit alterations relative to the parent GH9 endoglucanase in one ormore properties including: detergent stability (e.g. improved stabilityin a detergent composition,) and/or storage stability (e.g. improvedstorage stability in a detergent composition). Detergent compositionsmay include novel GH9 endoglucanase variants having activity on xanthangum pre-treated with xanthan lyase. Methods for producing and using thedetergent compositions are also disclosed. Variants as described hereinare particularly suitable for use in cleaning processes and detergentcompositions, such as laundry compositions and dish wash compositions,including hand wash and automatic dish wash compositions.

BACKGROUND

Xanthan gum is a polysaccharide derived from the bacterial coat ofXanthomonas campestris. It is produced by the fermentation of glucose,sucrose, or lactose by the Xanthomonas campestris bacterium. After afermentation period, the polysaccharide is precipitated from a growthmedium with isopropyl alcohol, dried, and ground into a fine powder.Later, it is added to a liquid medium to form the gum. Xanthan gum is anatural polysaccharide consisting of different sugars which areconnected by several different bonds, such asβ-D-mannosyl-β-D-1,4-glucuronosyl bonds andβ-D-glucosyl-β-D-1,4-glucosyl bonds. Xanthan gum is at least partlysoluble in water and forms highly viscous solutions or gels. Completeenzymatic degradation of xanthan gum requires several enzymaticactivities including xanthan lyase activity and endo-β-1,4-glucanaseactivity. Xanthan lyases are enzymes that cleave theβ-D-mannosyl-β-D-1,4-glucuronosyl bond of xanthan and have beendescribed in the literature. Xanthan lyases are known in the art, e.g.two xanthan lyases have been isolated from Paenibacillus alginolyticusXL-1 (e.g. Ruijssenaars et al. (1999) ‘A pyruvated mannose-specificxanthan lyase involved in xanthan degradation by Paenibacillusalginolyticus XL-1’, Appl. Environ. Microbiol. 65(6): 2446-2452, andRuijssenaars et al. (2000), ‘A novel gene encoding xanthan lyase ofPaenibacillus alginolyticus strain XL-1’, Appl. Environ. Microbiol.66(9): 3945-3950). Glycoside hydrolases are enzymes that catalyse thehydrolysis of the glycosyl bond to release smaller sugars. There areover 100 classes of glycoside hydrolases which have been classified, seeHenrissat et al. (1991) ‘A classification of glycosyl hydrolases basedon amino-acid sequence similarities’, J. Biochem. 280: 309-316 and theUniprot website at www.cazy.org. The glycoside hydrolase family 9 (GH9)consists of over 70 different enzymes that are mostly endo-glucanases(EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), β-glucosidases (EC3.2.1.21) and exo-β-glucosaminidase (EC 3.2.1.165). In recent yearsxanthan gum has been used as an ingredient in many consumer productsincluding foods (e.g. as thickening agent in salad dressings and dairyproducts) and cosmetics (e.g. as stabilizer and thickener in toothpasteand make-up, creams and lotions to prevent ingredients from separatingand to provide the right texture of the product). Further xanthan gumhas found use in the oil industry as an additive to regulate theviscosity of drilling fluids etc. The widespread use of xanthan gum hasled to a desire to degrade solutions, gels or mixtures containingxanthan gum thereby allowing easier removal of the byproducts.Endoglucanases and xanthan lyases for the degradation of xanthan gum andthe use of such enzymes for cleaning purposes, such as the removal ofxanthan gum containing stains, and in the drilling and oil industriesare known in the art, e.g. WO2013167581A1.

The known xanthan endoglucanase having SEQ ID NO: 2 was found to besensitive to storage in detergent. To improve the applicability and/orcost and/or the performance of such enzymes there is an ongoing searchfor variants with altered properties, such as increased stability, e.g.improved stability in a detergent composition. However, mutagenesis oflarge enzymes followed by purification and functional analysis of mutantlibraries can be very expensive and laborious.

SUMMARY

Since the known xanthan endoglucanase having SEQ ID NO: 2 is a largeenzyme (>1000 residues), it is difficult and expensive to randomlytarget its properties for improvement of, e.g., stability in a detergentcomposition.

In some aspects a non-limiting embodiment identifies regions in theprotein sequence/structure of the known xanthan endoglucanase having SEQID NO: 2 that are relevant for e.g. storage stability, and thereforeprovides an important guidance on where to mutate an endoglucanase inorder to stabilize the molecule in a detergent.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant, comprising analteration (e.g., a substitution, deletion or insertion) at one or morepositions in a region selected from the group consisting of: region 10corresponding to amino acids 1 to 94 of SEQ ID NO: 2, region 11corresponding to amino acids 106 to 114 of SEQ ID NO: 2, region 12corresponding to amino acids 139 to 209 of SEQ ID NO: 2, region 13corresponding to amino acids 252 to 266 of SEQ ID NO: 2, region 14corresponding to amino acids 302 to 338 of SEQ ID NO: 2, region 15corresponding to amino acids 362 to 546 of SEQ ID NO: 2, region 16corresponding to amino acids 596 to 611 of SEQ ID NO: 2, region 17corresponding to amino acids 661 to 805 of SEQ ID NO: 2, region 18corresponding to amino acids 829 to 838 of SEQ ID NO: 2, and region 19corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant, comprising analteration (e.g., a substitution, deletion or insertion) at one or morepositions in a region selected from the group consisting of: region 10corresponding to amino acids 1 to 94 of SEQ ID NO: 2, region 11corresponding to amino acids 106 to 114 of SEQ ID NO: 2, region 12corresponding to amino acids 139 to 209 of SEQ ID NO: 2, region 13corresponding to amino acids 252 to 266 of SEQ ID NO: 2, region 14corresponding to amino acids 302 to 338 of SEQ ID NO: 2, region 15corresponding to amino acids 362 to 546 of SEQ ID NO: 2, region 16corresponding to amino acids 596 to 611 of SEQ ID NO: 2, region 17corresponding to amino acids 661 to 805 of SEQ ID NO: 2, region 18corresponding to amino acids 829 to 838 of SEQ ID NO: 2, and region 19corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2, wherein saidvariant has at least 60% and less than 100% sequence identity to SEQ IDNO: 2; said endoglucanase variant has activity on xanthan gumpre-treated with xanthan lyase.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant having at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant, comprising analteration (e.g., a substitution, deletion or insertion) at one or morepositions in a region selected from the group consisting of:

-   i) region 10 corresponding to amino acids 1 to 94 of SEQ ID NO: 2,    e.g., said alteration at one or more positions selected from the    group consisting of positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,    12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,    29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,    46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,    63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,    80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, wherein    said positions correspond to amino acid positions of SEQ ID NO: 2    (e.g., using the numbering of SEQ ID NO: 2),-   ii) region 11 corresponding to amino acids 106 to 114 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 107, 108, 109, 110, 111, 112, 113,    114, wherein said positions correspond to amino acid positions of    SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO: 2),-   iii) region 12 corresponding to amino acids 139 to 209 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 139, 140, 141, 142, 143, 144, 145,    146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,    159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,    172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,    185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,    198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, wherein    said positions correspond to amino acid positions of SEQ ID NO: 2    (e.g., using the numbering of SEQ ID NO: 2),-   iv) region 13 corresponding to amino acids 252 to 266 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 252, 253, 254, 255, 256, 257, 258,    259, 260, 261, 262, 263, 264, 265, 266, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   v) region 14 corresponding to amino acids 302 to 338 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 302, 303, 304, 305, 306, 307, 308,    309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,    322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,    335, 336, 337, 338, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2),-   vi) region 15 corresponding to amino acids 362 to 546 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 362, 363, 364, 365, 366, 367, 368,    369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,    382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,    395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,    408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,    421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,    434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,    447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,    460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,    473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,    486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,    499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511,    512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,    525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,    538, 539, 540, 541, 542, 543, 544, 545, 546, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   vii) region 16 corresponding to amino acids 596 to 611 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 596, 597, 598, 599, 600, 601, 602,    603, 604, 605, 606, 607, 608, 609, 610, 611, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   viii) region 17 corresponding to amino acids 661 to 805 of SEQ ID    NO: 2, e.g., said alteration at one or more positions selected from    the group consisting of positions: 661, 662, 663, 664, 665, 666,    667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679,    680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,    693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705,    706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718,    719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731,    732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,    745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757,    758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770,    771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783,    784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796,    797, 798, 799, 800, 801, 802, 803, 804, 805, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   ix) region 18 corresponding to amino acids 829 to 838 to 1042 of SEQ    ID NO: 2, e.g., said alteration at one or more positions selected    from the group consisting of positions: 829, 830, 831, 832, 833,    834, 835, 836, 837, 838, wherein said positions correspond to amino    acid positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID    NO: 2)-   x) region 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions 1043, 1044, 1045, 1046, 1047, 1048,    1049, 1050, 1051, 1052, 1053, 1054, 1055, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2).-   In some aspects, a non-limiting embodiment relates to a detergent    composition comprising an endoglucanase variant, comprising an    alteration (e.g., a substitution, deletion or insertion) at one or    more positions in:-   a) one or more regions selected from the group consisting of:-   i) region 10 corresponding to amino acids 1 to 94 of SEQ ID NO: 2,    e.g., said alteration at one or more positions selected from the    group consisting of positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,    12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,    29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,    46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,    63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,    80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, wherein    said positions correspond to amino acid positions of SEQ ID NO: 2    (e.g., using the numbering of SEQ ID NO: 2),-   ii) region 11 corresponding to amino acids 106 to 114 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 107, 108, 109, 110, 111, 112, 113,    114, wherein said positions correspond to amino acid positions of    SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO: 2),-   iii) region 12 corresponding to amino acids 139 to 209 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 139, 140, 141, 142, 143, 144, 145,    146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,    159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,    172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,    185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,    198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, wherein    said positions correspond to amino acid positions of SEQ ID NO: 2    (e.g., using the numbering of SEQ ID NO: 2),-   iv) region 13 corresponding to amino acids 252 to 266 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 252, 253, 254, 255, 256, 257, 258,    259, 260, 261, 262, 263, 264, 265, 266, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   v) region 14 corresponding to amino acids 302 to 338 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 302, 303, 304, 305, 306, 307, 308,    309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,    322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,    335, 336, 337, 338, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2),-   vi) region 15 corresponding to amino acids 362 to 546 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 362, 363, 364, 365, 366, 367, 368,    369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,    382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,    395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,    408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,    421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,    434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,    447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,    460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,    473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,    486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,    499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511,    512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,    525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,    538, 539, 540, 541, 542, 543, 544, 545, 546, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   vii) region 16 corresponding to amino acids 596 to 611 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 596, 597, 598, 599, 600, 601, 602,    603, 604, 605, 606, 607, 608, 609, 610, 611, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   viii) region 17 corresponding to amino acids 661 to 805 of SEQ ID    NO: 2, e.g., said alteration at one or more positions selected from    the group consisting of positions: 661, 662, 663, 664, 665, 666,    667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679,    680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,    693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705,    706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718,    719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731,    732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,    745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757,    758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770,    771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783,    784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796,    797, 798, 799, 800, 801, 802, 803, 804, 805, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   ix) region 18 corresponding to amino acids 829 to 838 to 1042 of SEQ    ID NO: 2, e.g., said alteration at one or more positions selected    from the group consisting of positions: 829, 830, 831, 832, 833,    834, 835, 836, 837, 838, wherein said positions correspond to amino    acid positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID    NO: 2)-   x) region 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions 1043, 1044, 1045, 1046, 1047, 1048,    1049, 1050, 1051, 1052, 1053, 1054, 1055, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2).

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant having an alteration(e.g., a substitution, deletion or insertion) at one or more positionsselected from the group consisting of positions: 17, 20, 51, 53, 55, 56,60, 63, 79, 87, 186, 192, 302, 311, 313, 387, 388, 390, 403, 408, 410,416, 448, 451, 471, 472, 476, 489, 507, 512, 515, 538, 598, 599, 602,603, 605, 609, 676, 688, 690, 694, 697, 698, 699, 711, 719, 754, 756,760, 781, 786, 797, 833, 834, 835 and 1048 of SEQ ID NO: 2, whereinnumbering is according to SEQ ID NO: 2.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant having an alteration(e.g., a substitution, deletion or insertion) at one or more positionsselected from the group consisting of: 517A, F20P, F20N, F20G, F20Y,K51Q, K51H, E53P, E53G, Y55M, V56M, Y60F, S63F, T87R, A186P, K192N,1302D, 1302H, 1302V, 1302M, H311N, S313D, 1387T, K388R, K390Q, 1403Y,E408D, E408S, E408P, E408A, E408G, E408N, P410G, Q416S, Q416D, A448E,A448W, A448S, K451S, G471S, S472Y, D476R, Q489P, K507R, K512P, S515V,S538C, Y579W, S598Q, A599S, I602T, 1602D, V603P, 5605T, G609E, D676H,A688G, Y690F, T694A, T697G, R698W, T699A, T711V, T711Y, W719R, K754R,V756H, V756Y, 5760G, T781M, N786K, T797S, A824D, N833D, Q834E, S835D,and F1048W wherein numbering is according to SEQ ID NO: 2.

In one embodiment, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant comprising an alteration(e.g., a substitution, deletion or insertion) at one or more positionsselected from the group consisting of alterations in positions: 17, 20,302, 311, 313, 408, 476, 602, 688, 697, and 719, wherein numbering isaccording to SEQ ID NO: 2.

In an embodiment, the endoglucanase variant comprised in the detergentcompositions comprises an alteration at one or more positions selectedfrom the group consisting of alterations in positions: 517A, F20P,I302D, H311N, S313D, E408D, D476R, 1602T, A688G, T697G, and W719R,wherein numbering is according to SEQ ID NO: 2.

In an embodiment, the endoglucanase variant comprised in the detergentcompositions further comprises an alteration at one or more positionsselected from the group consisting of alterations in positions: 216,283, 346, 559, 564, 579, 636, 638, 651, 824, 848, 869, 880, 881, 883,887, 892, 905, 906, 912, 921, 928, 934, 937, 948, 956, 999, and 1037.

In an embodiment, the endoglucanase variant comprised in the detergentcompositions further comprises an alteration (e.g., substitution) at oneor more positions selected from the group consisting of: N216D, N216Q,A283D, A346D, A559P, A559N, A564E, Y579W, S636K, S636N, F638N, A651P,A824D, N848D, A869V, R880K, V881T, V881Q, T883R, T887K, T892P, N905D,F906A, A912V, K921R, S928D, Y934G, A937E, K948R, Q956Y, T999R, andA1037E.

In an embodiment, the endoglucanase variant comprised in the detergentcompositions comprises one or more of the following set ofsubstitutions:

F20Y + A448S + T781M E408D + K451S E408D + A448E E408D Y579W + E408D

In an embodiment, the endoglucanase variant comprised in the detergentcompositions comprises one or more of the following set ofsubstitutions:

S17A, F20P, N216D, A283D, H311N, E408D, Y579W, I602T, A651P, A688G,T883R, F906A, Y934G, Q956Y F20P, I302D, S313D, E408D, D476R, Y579W,S636K, T697G, V756Y, V881Q, T887K, F906A, A937E F20P, S313D, E408D,Y579W, S636K, A688G, T697G, N905D, A937E F20P, I302D, S313D, E408D,D476R, Y579W, S636K, T697G, W719R, V756Y, V881Q, T887K, F906A, A937EN216Q, S313D, E408D, D476R, Y579W, I602T, F638N, A651P, T697G, W719R,R880K, T887K, K921R, Y934G N216D, S313D, E408D, D476R, A564E, Y579W,I602T, F638N, A651P, Y690F, T697G, W719R, V756H, N833D, A869V, R880K,V881T, T887K, K921R, S928D, Y934G, T999R F20P, I302D, S313D, E408D,D476R, Y579W, S636K, T697G, W719R, V756Y, N848D, A869V, V881Q, T887K,N905D, F906A, Q912V, A937E, T999R, F1048W F20P, I302D, S313D, E408D,D476R, Q489P, Y579W, S636N, T697G, W719R, V756Y, A824D, N848D, V881Q,T887K, F906A, S928D, A937E F20P, I302D, S313D, E408D, Q416S, D476R,Q489P, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, A824D, N833D,N848D, T883R, T887K, F906A, A937E F20P, A186P, I302D, S313D, E408D,D476R, Q489P, Y579W, A599S, I602T, S636K, A651P, T697G, W719R, V756Y,N848D, T883R, T887K, F906A, A937E N216D, S313D, E408D, D476R, Y579W,I602T, V603P, F638N, A651P, A688G, T697G, W719R, V756H, R880K, T887K,K921R, S928D, Y934G, K948R F20P, K51Q, I302D, S313D, E408D, D476R,Q489P, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, N848D, T883R,T887K, F906A, A937E F20P, I302D, S313D, A346D, E408D, D476R, Q489P,Y579W, S636N, T697G, W719R, V756Y, A824D, N848D, V881Q, T887K, F906A,A937E, T999R F20P, I302D, S313D, E408D, D476R, Q489P, Y579W, I602T,S636N, T697G, W719R, V756Y, A824D, N848D, V881Q, T887K, N905D, F906A,A937E, T999R, A1037E, F1048W F20P, K51Q, I302D, S313D, E408D, D476R,Q489P, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, A824D, N848D,T883R, T887K, F906A, S928D, A937E, A1037E N216D, S313D, A346D, E408D,D476R, Q489P, A559P, Y579W, I602T, F638N, A651P, A688G, T697G, W719R,V756H, R880K, T887K, K921R, S928D, Y934G F20P, I302D, S313D, A346D,E408D, D476R, Q489P, Y579W, I602T, T697G, W719R, V756Y, N848D, V881Q,T887K, F906A, A937E N216D, S313D, E408D, D476R, Q489P, A559P, Y579W,I602T, F638N, A651P, A688G, T697G, W719R, V756H, Q834E, R880K, T887K,T892P, K921R, S928D, Y934G F20P, I302D, S313D, E408D, D476R, Q489P,A559N, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, N848D, T883R,T887K, F906A, S928D, A937E F20P, I302D, S313D, E408D, D476R, Q489P,Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, N848D, T883R, T887K,F906A, A937E F20P, I302D, S313D, E408D, Q416S, D476R, Q489P, A559N,Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, N848D, T883R, T887K,F906A, A937E F20P, I302D, S313D, E408D, D476R, Y579W, I602T, S636N,T697G, W719R, V756Y, A824D, N848D, V881Q, T887K, F906A, S928D, A937E,T999R, F1048W

In a particular aspect, the endoglucanase variant as described herein isone that does not comprise any amino acid alteration at a positionoutside of regions 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19. In thisaspect, the endoglucanase variant thus does not comprise any alteration(e.g., a substitution, deletion or insertion) in a region selected fromthe group consisting of: region 1 corresponding to amino acids 95 to 105of SEQ ID NO: 2, region 2 corresponding to amino acids 115 to 138 of SEQID NO: 2, region 3 corresponding to amino acids 210 to 251 of SEQ ID NO:2, region 4 corresponding to amino acids 267 to 301 of SEQ ID NO: 2,region 5 corresponding to amino acids 339 to 361 of SEQ ID NO: 2, region6 corresponding to amino acids 547 to 595 of SEQ ID NO: 2, region 7corresponding to amino acids 612 to 660 of SEQ ID NO: 2, region 8corresponding to amino acids 806 to 828 of SEQ ID NO: 2, and region 9corresponding to amino acids 839 to 1045 of SEQ ID NO: 2.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant having activity onxanthan gum pre-treated with xanthan lyase; said activity comprisesendoglucanase EC 3.2.1.4 activity, said activity is endoglucanase EC3.2.1.4 activity.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant having an improvedstability in a detergent composition compared to a parent endoglucanase(e.g., with SEQ ID NO: 2).

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an endoglucanase variant having a half-lifeimprovement factor (HIF) of >1.0 relative to a parent endoglucanase,e.g. an endoglucanase of SEQ ID NO: 2.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising an isolated GH9 endoglucanase variant havingactivity on xanthan gum pre-treated with xanthan lyase. In a furtheraspect, the detergent composition further comprises an isolatedpolypeptide having xanthan lyase activity.

In some aspects, a non-limiting embodiment relates to a detergentcomposition comprising one or more detergent components for degradingxanthan gum.

In some aspects, a non-limiting embodiment relates to use of a detergentcomposition, wherein said use is selected from the group consisting of:use for degrading xanthan gum and use in a cleaning process, such aslaundry or hard surface cleaning such as dish wash.

In some aspects, a non-limiting embodiment further relates to the use ofa detergent composition for degrading xanthan gum, for washing orcleaning textiles and/or hard surfaces, such as dish wash, wherein thecomposition has an enzyme detergency benefit.

In some aspects, a non-limiting embodiment also relates to methods ofdegrading xanthan gum using detergent compositions, wherein xanthan gumis on the surface of a hard surface or textile.

Overview of Sequence Listing

SEQ ID NO: 1 is the DNA sequence of the parent mature endoglucanase froma strain of a Paenibacillus sp.

SEQ ID NO: 2 is the amino acid sequence of mature polypeptide encoded bySEQ ID NO: 1.

SEQ ID NO: 3 is the DNA sequence of the alpha-amylase secretion signalfrom Bacillus licheniformis.

SEQ ID NO: 4 is the amino acid sequence of the alpha-amylase secretionsignal from Bacillus licheniformis.

Definitions

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cleaning or Detergent Application: the term “cleaning or detergentapplication” means applying the endoglucanase of the application in anycomposition for the purpose of cleaning or washing, by hand, machine orautomated, a hard surface or a textile.

Cleaning Composition: the term “cleaning composition” refers tocompositions that find use in the removal of undesired compounds fromitems to be cleaned, such as textiles, dishes, and hard surfaces. Theterms encompass any materials/compounds selected for the particular typeof cleaning composition desired and the form of the product (e.g.,liquid, gel, powder, granulate, paste, or spray compositions) andincludes, but is not limited to, detergent compositions (e.g., liquidand/or solid laundry detergents and fine fabric detergents; hard surfacecleaning formulations, such as for glass, wood, ceramic and metalcounter tops and windows; carpet cleaners; oven cleaners; fabricfresheners; fabric softeners; and textile and laundry pre-spotters, aswell as dish wash detergents). In addition to the endoglucanase, thedetergent formulation may contain one or more additional enzymes (suchas xanthan lyases, proteases, amylases, lipases, cutinases, cellulases,endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidases, haloperoxygenases, catalases and mannanases, or any mixturethereof), and/or components such as surfactants, builders, chelators orchelating agents, bleach system or bleach components, polymers, fabricconditioners, foam boosters, suds suppressors, dyes, perfume, tannishinhibitors, optical brighteners, bactericides, fungicides, soilsuspending agents, anti-corrosion agents, enzyme inhibitors orstabilizers, enzyme activators, transferase(s), hydrolytic enzymes,oxido reductases, bluing agents and fluorescent dyes, antioxidants, andsolubilizers.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Colour clarification: During washing and wearing loose or broken fiberscan accumulate on the surface of the fabrics. One consequence can bethat the colours of the fabric appear less bright or less intensebecause of the surface contaminations. Removal of the loose or brokenfibers from the textile will partly restore the original colours andlooks of the textile. By the term “colour clarification”, as usedherein, is meant the partial restoration of the initial colours oftextile.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide. Each control sequence may be native (La, from the samegene) or foreign (i.e., from a different gene) to the polynucleotideencoding the polypeptide or native or foreign to each other. Suchcontrol sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a polypeptide.

Corresponding to: The term “corresponding to” as used herein, refers toa way of determining the specific amino acid of a sequence whereinreference is made to a specific amino acid sequence. E.g. for thepurposes, when references are made to specific amino acid positions, theskilled person would be able to align another amino acid sequence tosaid amino acid sequence that reference has been made to, in order todetermine which specific amino acid may be of interest in said anotheramino acid sequence. Alignment of another amino acid sequence with e.g.the sequence as set forth in SEQ ID NO: 2, or any other sequence listedherein, has been described elsewhere herein. Alternative alignmentmethods may be used, and are well-known for the skilled person.

Degrading xanthan gum and xanthan gum degrading activity: The terms“degrading xanthan gum” and “xanthan gum degrading activity” are usedinterchangeably and are defined as the depolymerization, degradation orbreaking down of xanthan gum into smaller components. The degradation ofxanthan gum can either be the removal of one or more side chainsaccharides, the cutting of the backbone of xanthan gum into smallercomponents or the removal of one or more side chain saccharides and thecutting of the backbone of xanthan gum into smaller components. Anon-limiting assay for measuring degradation of xanthan gum is thereducing sugar assay as described in example 3 herein. Non-limitingexamples of the xanthan gum degrading activity include endoglucanase EC3.2.1.4 activity.

Detergent component: the term “detergent component” is defined herein tomean the types of chemicals which can be used in detergent compositions.Examples of detergent components are surfactants, hydrotropes, builders,co-builders, chelators or chelating agents, bleaching system or bleachcomponents, polymers, fabric hueing agents, fabric conditioners, foamboosters, suds suppressors, dispersants, dye transfer inhibitors,fluorescent whitening agents, perfume, optical brighteners,bactericides, fungicides, soil suspending agents, soil release polymers,anti-redeposition agents, enzyme inhibitors or stabilizers, enzymeactivators, antioxidants, and solubilizers. The detergent compositionmay comprise of one or more of any type of detergent component.

Detergent composition: the term “detergent composition” refers tocompositions that find use in the removal of undesired compounds fromitems to be cleaned, such as textiles, dishes, and hard surfaces. Thedetergent composition may be used to e.g. clean textiles, dishes andhard surfaces for both household cleaning and industrial cleaning. Theterms encompass any materials/compounds selected for the particular typeof cleaning composition desired and the form of the product (e.g.,liquid, gel, powder, granulate, paste, or spray compositions) andincludes, but is not limited to, detergent compositions (e.g., liquidand/or solid laundry detergents and fine fabric detergents; hard surfacecleaning formulations, such as for glass, wood, ceramic and metalcounter tops and windows; carpet cleaners; oven cleaners; fabricfresheners; fabric softeners; and textile and laundry pre-spotters, aswell as dish wash detergents). In addition to containing a GH9endoglucanase as described herein and/or xanthan lyase, the detergentformulation may contain one or more additional enzymes (such as xanthanlyases, proteases, amylases, lipases, cutinases, cellulases,endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidases, haloperoxygenases, catalases and mannanases, or any mixturethereof), and/or components such as surfactants, builders, chelators orchelating agents, bleach system or bleach components, polymers, fabricconditioners, foam boosters, suds suppressors, dyes, perfume, tannishinhibitors, optical brighteners, bactericides, fungicides, soilsuspending agents, anti-corrosion agents, enzyme inhibitors orstabilizers, enzyme activators, transfer-ase(s), hydrolytic enzymes,oxido reductases, bluing agents and fluorescent dyes, antioxidants, andsolubilizers.

Dish wash: The term “dish wash” refers to all forms of washing dishes,e.g. by hand or automatic dish wash. Washing dishes includes, but is notlimited to, the cleaning of all forms of crockery such as plates, cups,glasses, bowls, all forms of cutlery such as spoons, knives, forks andserving utensils as well as ceramics, plastics, metals, china, glass andacrylics.

Dish washing composition: The term “dish washing composition” refers toall forms of compositions for cleaning hard surfaces. The presentinvention is not restricted to any particular type of dish washcomposition or any particular detergent.

Endoglucanase: The term “endoglucanase” or “EG” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans, xyloglucans, xanthans and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). A non-limiting assay for measuringendoglucanase activity is the reducing sugar assay as described inexample 3 herein. Non-limiting examples of endoglucanases include themature parent endoglucanase having SEQ ID NO: 2.

Enzyme detergency benefit: The term “enzyme detergency benefit” isdefined herein as the advan-tageous effect an enzyme may add to adetergent compared to the same detergent without the enzyme. Importantdetergency benefits which can be provided by enzymes are stain removalwith no or very little visible soils after washing and or cleaning,prevention or reduction of redeposition of soils released in the washingprocess an effect that also is termed anti-redeposition, restoring fullyor partly the whiteness of textiles, which originally were white butafter repeated use and wash have obtained a greyish or yellowishappearance an effect that also is termed whitening. Textile carebenefits, which are not directly related to catalytic stain removal orprevention of redeposition of soils are also important for enzymedetergency benefits. Examples of such textile care benefits areprevention or reduction of dye transfer from one fabric to anotherfabric or another part of the same fabric an effect that is also termeddye transfer inhibition or anti-backstaining, removal of protruding orbroken fibers from a fabric surface to decrease pilling tendencies orremove already existing pills or fuzz an effect that also is termedanti-pilling, improvement of the fabric-softness, colour clarificationof the fabric and removal of particulate soils which are trapped in thefibers of the fabric or garment. Enzymatic bleaching is a further enzymedetergency benefit where the catalytic activity generally is used tocatalyze the formation of bleaching component such as hydrogen peroxideor other peroxides.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has endoglucanaseactivity. In one aspect, a fragment contains at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% of the number of amino acids ofthe mature polypeptide.

Endoglucanase variant having activity on xanthan gum pre-treated withxanthan lyase: The term “Endoglucanase variant having activity onxanthan gum pre-treated with xanthan lyase” or an “endoglucanase havingactivity on xanthan gum pre-treated with xanthan lyase and belonging tothe GH9 class of glycosyl hydrolases” is defined as a polypeptidecomprising a domain belonging to the GH9 class of glycosyl hydrolases,and having activity (e.g. enzymatic activity, xanthan degradingactivity, endoglucanase EC 3.2.1.4 activity) on xanthan gum pre-treatedwith xanthan lyase. A non-limiting assay for measuring activity onxanthan gum pre-treated with xanthan lyase is disclosed in example 3herein.

Xanthan lyase variant having activity on xanthan gum: The term “Xanthanlyase variant having activity on xanthan gum” is defined as apolypeptide that cleaves the β-D-mannosyl-β-D-1,4-glucuronosyl bond ofxanthan (e.g., xanthan lyase EC 4.2.2.12 activity). Examples of thexanthan lyase variants having activity on xanthan gum, are xanthan lyasepolypeptides as such. Thus, polypeptides that that cleaves theβ-D-mannosyl-β-D-1,4-glucuronosyl bond of xanthan.

Half-life: the term “half-life” is the time it takes for an enzyme tolose half of its enzymatic activity under a given set of conditions. Itis denoted as T % and is measured in hours (hrs). Half-lives can becalculated at a given detergent concentration and storage temperaturefor parent (e.g. wild-type) and/or variants, as the degradation followsan exponential decay and the incubation time (hours) is known, i.e.,according to the following formulas:

T1/2(variant)=(Ln(0.5)/Ln(RA-variant/100))*Time

T1/2(wild-type)=(Ln(0.5)/Ln(RA-wild-type/100))*Time

Where ‘RA’ is the residual activity in percent and ‘Time’ is theincubation time in hours.

Half-life improvement factor: the term “Half-life improvement factor” or“HIF” is the improvement of half-life of a variant compared to theparent polypeptide, such as the parent endoglucanase. A half-lifeimprovement factor (HIF) under a given set of storage conditions(detergent concentration and temperature) can be calculated as:

${HIF} = \frac{{T\; 1\text{/}2},{variant}}{{T\; 1\text{/}2},{wt}}$

where the wild-type (wt) is incubated under the same storage condition(detergent concentration and incubation temperature) as the variant. Inthe cases where the difference in stability between wild-type andvariant is too big to accurately assess half-life for both wild-type andvariant using the same incubation time, the incubation time forwild-type and variant is different e.g. 1 h for wild-type and 138 h forthe most stable variants.

A non-limiting way of calculating HIF is also described in example 3herein.

Hard surface cleaning: The term “Hard surface cleaning” is definedherein as cleaning of hard surfaces wherein hard surfaces may includefloors, tables, walls, roofs etc. as well as surfaces of hard objectssuch as cars (car wash) and dishes (dish wash). Dish washing includesbut are not limited to cleaning of plates, cups, glasses, bowls, andcutlery such as spoons, knives, forks, serving utensils, ceramics,plastics, metals, china, glass and acrylics.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotide.The term “host cell” encompasses any progeny of a parent cell that isnot identical to the parent cell due to mutations that occur duringreplication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to, catalyticefficiency, catalytic rate, chemical stability, oxidation stability, pHactivity, pH stability, specific activity, stability under storageconditions, chelator stability, substrate binding, substrate cleavage,substrate specificity, substrate stability, surface properties, thermalactivity, and thermostability.

Improved wash performance: The term “improved wash performance” isdefined herein as a (variant) enzyme (also a blend of enzymes, notnecessarily only variants but also backbones, and in combination withcertain cleaning composition etc.) displaying an alteration of the washperformance of a variant relative to the wash performance of the parentvariant e.g. by increased stain removal. The term “wash performance”includes wash performance in laundry but also e.g. in dish wash.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Laundering: The term “laundering” relates to both household launderingand industrial laundering and means the process of treating textileswith a solution containing a cleaning or detergent composition. Thelaundering process can for example be carried out using e.g. a householdor an industrial washing machine or can be carried out by hand.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 1055 of SEQ ID NO: 2.

It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide. Itis also known in the art that different host cells process polypeptidesdifferently, and thus, one host cell expressing a polynucleotide mayproduce a different mature polypeptide (e.g., having a differentC-terminal and/or N-terminal amino acid) as compared to another hostcell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving enzymatic activity such as activity on xanthan gum pre-treatedwith xanthan lyase or xanthan lyase activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 1 to 3165 of SEQ ID NO: 1.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent: The term “parent” or “parent endoglucanase” means anypolypeptide with endoglucanase activity to which an alteration is madeto produce the enzyme variants. In one aspect, the parent is anendoglucanase having the identical amino acid sequence of the variant,but not having the alterations at one or more of the specifiedpositions. It will be understood, that the expression “having identicalamino acid sequence” relates to 100% sequence identity. Non-limitingexamples of parent endoglucanases include the mature parentendoglucanase having SEQ ID NO: 2.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”. For purposes, the sequence identity between two amino acidsequences is determined using the Needleman-Wunsch algorithm (Needlemanand Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), such as version 5.0.0 or later. The parameters used are gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes, the sequence identity between two deoxyribonucleotidesequences is determined using the Needleman-Wunsch algorithm (Needlemanand Wunsch, 1970, supra) as implemented in the Needle program of theEMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, supra), such as version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitutionmatrix. The output of Needle labeled “longest identity” (obtained usingthe −nobrief option) is used as the percent identity and is calculatedas follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stringency conditions: The different stringency conditions are definedas follows.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 25% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having enzymatic activity, such as activity on xanthan gumpre-treated with xanthan lyase or xanthan lyase activity.

Textile: The term “textile” means any textile material including yarns,yarn intermediates, fibers, non-woven materials, natural materials,synthetic materials, and any other textile material, fabrics made ofthese materials and products made from fabrics (e.g., garments and otherarticles). The textile or fabric may be in the form of knits, wovens,denims, non-wovens, felts, yarns, and towelling. The textile may becellulose based such as natural cellulosics, including cotton,flax/linen, jute, ramie, sisal or coir or manmade cellulosics (e.g.originating from wood pulp) including viscose/rayon, ramie, celluloseacetate fibers (tricell), lyocell or blends thereof. The textile orfabric may also be non-cellulose based such as natural polyamidesincluding wool, camel, cashmere, mohair, rabbit and silk or syntheticpolymer such as nylon, aramid, polyester, acrylic, polypropylene andspandex/elastane, or blends thereof as well as blend of cellulose basedand non-cellulose based fibers. Examples of blends are blends of cottonand/or rayon/viscose with one or more companion material such as wool,synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyesterfibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyurethane fibers, polyurea fibers, aramid fibers), andcellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen,jute, cellulose acetate fibers, lyocell). Fabric may be conventionalwashable laundry, for example stained household laundry. When the termfabric or garment is used it is intended to include the broader termtextiles as well.

Textile care benefit: “Textile care benefits”, which are not directlyrelated to catalytic stain removal or prevention of redeposition ofsoils, are also important for enzyme detergency benefits. Examples ofsuch textile care benefits are prevention or reduction of dye transferfrom one textile to another textile or another part of the same textilean effect that is also termed dye transfer inhibition oranti-backstaining, removal of protruding or broken fibers from a textilesurface to decrease pilling tendencies or remove already existing pillsor fuzz an effect that also is termed anti-pilling, improvement of thetextile-softness, colour clarification of the textile and removal ofparticulate soils which are trapped in the fibers of the textile.Enzymatic bleaching is a further enzyme detergency benefit where thecatalytic activity generally is used to catalyze the formation ofbleaching component such as hydrogen peroxide or other peroxides orother bleaching species.

Variant: The term “variant” means a polypeptide (e.g., a GH9endoglucanase polypeptide) comprising an alteration i.e., asubstitution, insertion, and/or deletion, at one or more (e.g., several)positions. A substitution means replacement of the amino acid occupyinga position with a different amino acid; a deletion means removal of theamino acid occupying a position; and an insertion means adding one ormore (e.g. several) amino acids e.g., 1-5 amino acids adjacent to andimmediately following the amino acid occupying a position. Non-limitingexamples of endoglucanase variants as described herein includeendoglucanase variants having an activity on xanthan gum pre-treatedwith xanthan lyase. Non-limiting examples of variants as describedherein further include variants having at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% endoglucanase activity of the mature parenthaving SEQ ID NO: 2. A non-limiting assay for measuring activity onxanthan gum pre-treated with xanthan lyase is disclosed in example 3herein.

Stability: The term “stability” means resistance or the degree ofresistance to change, unfolding, disintegration, denaturation oractivity loss. Non-limiting examples of stability include conformationalstability, storage stability and stability during use, e.g. during awash process and reflects the stability of a polypeptide (e.g. anendoglucanase variant) as a function of time, e.g. how much activity isretained when said polypeptide (e.g. said endoglucanase variant) is keptin solution, in particular in a detergent solution. The stability isinfluenced by many factors, e.g. presence of chelator(s), pH,temperature, detergent composition, e.g. amount of builders,surfactants, chelators etc. The endoglucanase stability may be measuredusing a half-life improvement factor (HIF) as described in example 3herein. The endoglucanase stability may also be measured using areducing sugar assay as described in example 3 herein.

Improved stability: The term “improved stability” or “increasedstability” is defined herein as increased stability in a detergentcomposition (e.g., in solutions), relative to the stability of theparent endoglucanase, relative to an endoglucanase having the identicalamino acid sequence of the variant, but not having the alterations atone or more of the specified positions, or relative to SEQ ID NO: 2. Theterms “improved stability” and “increased stability” includes “improvedchemical stability”, “detergent stability” and “improved detergentstability.

Improved chemical stability: The term “improved chemical stability” isdefined herein as a variant enzyme displaying retention of enzymaticactivity after a period of incubation in the presence of a chemical orchemicals, either naturally occurring or synthetic, which reduces theenzymatic activity of the parent enzyme. Improved chemical stability mayalso result in variants being more able (e.g., better that the parent)to catalyze a reaction in the presence of such chemicals. In aparticular aspect the improved chemical stability is an improvedstability in a detergent, in particular in a liquid detergent. The term“detergent stability” or “improved detergent stability is in particularan improved stability of the endoglucanase compared to the parentendoglucanase, when an endoglucanase variant as described herein ismixed into a liquid detergent formulation, especially into a liquiddetergent formulation comprising a chelator (e.g. EDTA or citrate).

Conformational stability: The term “conformational stability” meansresistance or a degree of resistance to conformational change, unfoldingor disintegration. Accordingly, the term “less conformationally stable”means less resistant or having lesser degree of resistance toconformational change, unfolding or disintegration.

Instability: The term “instability” means lack of stability.Non-limiting examples of instability include conformational instability,unfolding, denaturation, disintegration, activity loss.

Wash performance: The term “wash performance” is used as an enzyme'sability to remove stains present on the object to be cleaned during e.g.wash or hard surface cleaning. The improvement in the wash performancemay be quantified by calculating the so-called intensity value (Int) in‘Automatic Mechanical Stress Assay (AMSA) for laundry’ or the remissionvalue (Rem) as defined herein.

Conventions for Designation of Variants

For purposes, the mature polypeptide disclosed in SEQ ID NO: 2 is usedto determine the corresponding amino acid residue in anotherendoglucanase. The amino acid sequence of another endoglucanase isaligned with the mature polypeptide disclosed in SEQ ID NO: 2, and basedon the alignment, the amino acid position number corresponding to anyamino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2is determined using the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleprogram of the EMBOSS package (EMBOSS: The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),version 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version ofBLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherendoglucanase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Altschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample, the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implemen-tation of thesealgorithms can additionally be utilized to query structure databaseswith a structure of interest in order to discover possible structuralhomologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants, the nomenclature described below is adaptedfor ease of reference. The accepted IUPAC single letter or three letteramino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly, the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.Multiple alteration variants may also be separated by a comma (“,”),e.g., “Arg170Tyr,Gly195Glu” or “R170Y,G195E” representing a substitutionof arginine and glycine at positions 170 and 195 with tyrosine andglutamic acid, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION

The known xanthan endoglucanase having SEQ ID NO: 2 is a large enzyme(>1000 residues), it is therefore extremely laborious and expensive totarget its properties for improvement of, e.g. stability in a detergentcomposition. In some aspects, a non-limiting embodiment has targetedspecific regions of the endoglucanase which have an effect on thestability of the endoglucanase molecules using protein engineering to aregion selected from the group consisting of: region 10 corresponding toamino acids 1 to 94 of SEQ ID NO: 2, region 11 corresponding to aminoacids 106 to 114 of SEQ ID NO: 2, region 12 corresponding to amino acids139 to 209 of SEQ ID NO: 2, region 13 corresponding to amino acids 252to 266 of SEQ ID NO: 2, region 14 corresponding to amino acids 302 to338 of SEQ ID NO: 2, region 15 corresponding to amino acids 362 to 546of SEQ ID NO: 2, region 16 corresponding to amino acids 596 to 611 ofSEQ ID NO: 2, region 17 corresponding to amino acids 661 to 805 of SEQID NO: 2, region 18 corresponding to amino acids 829 to 838 of SEQ IDNO: 2, and region 19 corresponding to amino acids 1043 to 1055 of SEQ IDNO: 2.

In one embodiment the detergent compositions dramatically decrease thenumber of residues to target when trying to stabilize endoglucanasemolecules using protein engineering.

Variants

In one embodiment, regions in the protein sequence of the known xanthanendoglucanase having SEQ ID NO: 2 that are affected when the molecule isincubated in detergent, are the following: region 10 corresponding toamino acids 1 to 94 of SEQ ID NO: 2, region 11 corresponding to aminoacids 106 to 114 of SEQ ID NO: 2, region 12 corresponding to amino acids139 to 209 of SEQ ID NO: 2, region 13 corresponding to amino acids 252to 266 of SEQ ID NO: 2, region 14 corresponding to amino acids 302 to338 of SEQ ID NO: 2, region 15 corresponding to amino acids 362 to 546of SEQ ID NO: 2, region 16 corresponding to amino acids 596 to 611 ofSEQ ID NO: 2, region 17 corresponding to amino acids 661 to 805 of SEQID NO: 2, region 18 corresponding to amino acids 829 to 838 of SEQ IDNO: 2, and region 19 corresponding to amino acids 1043 to 1055 of SEQ IDNO: 2. This embodiment relates to an important guidance on where tomutate an endoglucanase in order to stabilize its molecule in adetergent.

In one embodiment a detergent composition may include an endoglucanasevariant, comprising an alteration (e.g., a substitution, deletion orinsertion) at one or more positions in a region selected from the groupconsisting of: region 10 corresponding to amino acids 1 to 94 of SEQ IDNO: 2, region 11 corresponding to amino acids 106 to 114 of SEQ ID NO:2, region 12 corresponding to amino acids 139 to 209 of SEQ ID NO: 2,region 13 corresponding to amino acids 252 to 266 of SEQ ID NO: 2,region 14 corresponding to amino acids 302 to 338 of SEQ ID NO: 2,region 15 corresponding to amino acids 362 to 546 of SEQ ID NO: 2,region 16 corresponding to amino acids 596 to 611 of SEQ ID NO: 2,region 17 corresponding to amino acids 661 to 805 of SEQ ID NO: 2,region 18 corresponding to amino acids 829 to 838 of SEQ ID NO: 2, andregion 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2;said endoglucanase variant has activity on xanthan gum pre-treated withxanthan lyase, said activity is a xanthan gum degrading activity.

In one embodiment, a detergent composition may include an endoglucanasevariant, comprising an alteration (e.g., a substitution, deletion orinsertion) at one or more positions in a region selected from the groupconsisting of:

-   i) region 10 corresponding to amino acids 1 to 94 of SEQ ID NO: 2,    e.g., said alteration at one or more positions selected from the    group consisting of positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,    12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,    29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,    46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,    63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,    80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, wherein    said positions correspond to amino acid positions of SEQ ID NO: 2    (e.g., using the numbering of SEQ ID NO: 2),-   ii) region 11 corresponding to amino acids 106 to 114 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 107, 108, 109, 110, 111, 112, 113,    114, wherein said positions correspond to amino acid positions of    SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO: 2),-   iii) region 12 corresponding to amino acids 139 to 209 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 139, 140, 141, 142, 143, 144, 145,    146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,    159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,    172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,    185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,    198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, wherein    said positions correspond to amino acid positions of SEQ ID NO: 2    (e.g., using the numbering of SEQ ID NO: 2),-   iv) region 13 corresponding to amino acids 252 to 266 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 252, 253, 254, 255, 256, 257, 258,    259, 260, 261, 262, 263, 264, 265, 266, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   v) region 14 corresponding to amino acids 302 to 338 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 302, 303, 304, 305, 306, 307, 308,    309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,    322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,    335, 336, 337, 338, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2),-   vi) region 15 corresponding to amino acids 362 to 546 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 362, 363, 364, 365, 366, 367, 368,    369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,    382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,    395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,    408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,    421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,    434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,    447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,    460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,    473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,    486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,    499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511,    512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,    525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,    538, 539, 540, 541, 542, 543, 544, 545, 546, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   vii) region 16 corresponding to amino acids 596 to 611 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 596, 597, 598, 599, 600, 601, 602,    603, 604, 605, 606, 607, 608, 609, 610, 611, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   viii) region 17 corresponding to amino acids 661 to 805 of SEQ ID    NO: 2, e.g., said alteration at one or more positions selected from    the group consisting of positions: 661, 662, 663, 664, 665, 666,    667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679,    680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,    693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705,    706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718,    719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731,    732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,    745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757,    758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770,    771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783,    784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796,    797, 798, 799, 800, 801, 802, 803, 804, 805, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   ix) region 18 corresponding to amino acids 829 to 838 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 829, 830, 831, 832, 833, 834, 835,    836, 837, 838, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2)-   x) region 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions 1043, 1044, 1045, 1046, 1047, 1048,    1049, 1050, 1051, 1052, 1053, 1054, 1055, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2).

In one embodiment a detergent composition may include an endoglucanasevariant, comprising an alteration (e.g., a substitution, deletion orinsertion) at one or more positions in one or more regions selected fromthe group consisting of: region 10 corresponding to amino acids 1 to 94of SEQ ID NO: 2, region 11 corresponding to amino acids 106 to 114 ofSEQ ID NO: 2, region 12 corresponding to amino acids 139 to 209 of SEQID NO: 2, region 13 corresponding to amino acids 252 to 266 of SEQ IDNO: 2, region 14 corresponding to amino acids 302 to 338 of SEQ ID NO:2, region 15 corresponding to amino acids 362 to 546 of SEQ ID NO: 2,region 16 corresponding to amino acids 596 to 611 of SEQ ID NO: 2,region 17 corresponding to amino acids 661 to 805 of SEQ ID NO: 2,region 18 corresponding to amino acids 829 to 838 of SEQ ID NO: 2, andregion 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2,wherein said variant has at least 60% and less than 100% sequenceidentity to SEQ ID NO: 2; said endoglucanase variant has activity onxanthan gum pre-treated with xanthan lyase, said activity is a xanthangum degrading activity.

In one embodiment a detergent composition may include an endoglucanasevariant as described herein having multiple alterations (such as 2, 3,4, 5, 6, 7, 8, 9 or 10) in one region (e.g., of SEQ ID NO: 2 or anotherparent endoglucanase) selected from the group consisting of: region 10corresponding to amino acids 1 to 94 of SEQ ID NO: 2, region 11corresponding to amino acids 106 to 114 of SEQ ID NO: 2, region 12corresponding to amino acids 139 to 209 of SEQ ID NO: 2, region 13corresponding to amino acids 252 to 266 of SEQ ID NO: 2, region 14corresponding to amino acids 302 to 338 of SEQ ID NO: 2, region 15corresponding to amino acids 362 to 546 of SEQ ID NO: 2, region 16corresponding to amino acids 596 to 611 of SEQ ID NO: 2, region 17corresponding to amino acids 661 to 805 of SEQ ID NO: 2, region 18corresponding to amino acids 829 to 838 of SEQ ID NO: 2, and region 19corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2, wherein saidvariant has at least 60% and less than 100% sequence identity to SEQ IDNO: 2, said endoglucanase variant has activity on xanthan gumpre-treated with xanthan lyase, said activity is a xanthan gum degradingactivity.

In one embodiment a detergent composition may include an endoglucanasevariant as described herein having multiple alterations (e.g., 2, 3, 4,5, 6, 7, 8, 9 or 10) in multiple regions (e.g., 2, 3, 4, 5, 6, 7, 8, 9or 10) (e.g., of SEQ ID NO: 2 or another parent endoglucanase) selectedfrom the group consisting of: region 10 corresponding to amino acids 1to 94 of SEQ ID NO: 2, region 11 corresponding to amino acids 106 to 114of SEQ ID NO: 2, region 12 corresponding to amino acids 139 to 209 ofSEQ ID NO: 2, region 13 corresponding to amino acids 252 to 266 of SEQID NO: 2, region 14 corresponding to amino acids 302 to 338 of SEQ IDNO: 2, region 15 corresponding to amino acids 362 to 546 of SEQ ID NO:2, region 16 corresponding to amino acids 596 to 611 of SEQ ID NO: 2,region 17 corresponding to amino acids 661 to 805 of SEQ ID NO: 2,region 18 corresponding to amino acids 829 to 838 of SEQ ID NO: 2, andregion 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2,said endoglucanase variant has activity on xanthan gum pre-treated withxanthan lyase, said activity is a xanthan gum degrading activity.

In one embodiment, detergent compositions may include endoglucanasevariants, comprising an alteration (e.g., a substitution, deletion orinsertion) at one or more (e.g., several) positions of the mature parentpolypeptide (e.g., SEQ ID NO: 2), wherein each alteration isindependently a substitution, insertion or deletion, wherein the varianthas endoglucanase activity.

In an embodiment, the variant has sequence identity of at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, to the amino acid sequence of the parentendoglucanase.

In one embodiment, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the mature polypeptide of SEQ ID NO: 2.

In one embodiment a detergent composition may include an endoglucanasevariant, wherein said variant has at least 61%, at least 62%, at least63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to SEQ ID NO: 2.

In another aspect, a variant comprises an alteration at one or more(e.g., several) positions corresponding to positions 17, 20, 51, 53, 55,56, 60, 63, 79, 87, 186, 192, 302, 311, 313, 387, 388, 390, 403, 408,410, 416, 448, 451, 471, 472, 476, 489, 507, 512, 515, 538, 598, 599,602, 603, 605, 609, 676, 688, 690, 694, 697, 698, 699, 711, 719, 754,756, 760, 781, 786, 797, 833, 834, 835 and 1048 of SEQ ID NO: 2, whereinnumbering is according to SEQ ID NO: 2.

In one embodiment, the endoglucanase variant comprised in the detergentcompositions further comprises an alteration at one or more positionsselected from the group consisting of alterations in positions: 216,283, 346, 559, 564, 579, 636, 638, 651, 824, 848, 869, 880, 881, 883,887, 892, 905, 906, 912, 921, 928, 934, 937, 948, 956, 999 and 1037 ofSEQ ID NO: 2.

In another aspect, a variant comprises an alteration at two positionscorresponding to any of positions 17, 20, 51, 53, 55, 56, 60, 63, 79,87, 186, 192, 302, 311, 313, 387, 388, 390, 403, 408, 410, 416, 448,451, 471, 472, 476, 489, 507, 512, 515, 538, 598, 599, 602, 603, 605,609, 676, 688, 690, 694, 697, 698, 699, 711, 719, 754, 756, 760, 781,786, 797, 833, 834, 835 and 1048 of SEQ ID NO: 2, wherein numbering isaccording to SEQ ID NO: 2.

In one embodiment, the endoglucanase variant comprised in the detergentcompositions further comprises an alteration at one or more positionsselected from the group consisting of alterations in positions: 216,283, 346, 559, 564, 579, 636, 638, 651, 824, 848, 869, 880, 881, 883,887, 892, 905, 906, 912, 921, 928, 934, 937, 948, 956, 999 and 1037 ofSEQ ID NO: 2.

In another aspect, a variant comprises an alteration at three positionscorresponding to any of positions 17, 20, 51, 53, 55, 56, 60, 63, 79,87, 186, 192, 302, 311, 313, 387, 388, 390, 403, 408, 410, 416, 448,451, 471, 472, 476, 489, 507, 512, 515, 538, 598, 599, 602, 603, 605,609, 676, 688, 690, 694, 697, 698, 699, 711, 719, 754, 756, 760, 781,786, 797, 833, 834, 835 and 1048 of SEQ ID NO: 2, wherein numbering isaccording to SEQ ID NO: 2.

In one embodiment, the endoglucanase variant comprised in the detergentcompositions further comprises an alteration at one or more positionsselected from the group consisting of alterations in positions: 216,283, 346, 559, 564, 579, 636, 638, 651, 824, 848, 869, 880, 881, 883,887, 892, 905, 906, 912, 921, 928, 934, 937, 948, 956, 999 and 1037 ofSEQ ID NO: 2.

Thus, in one embodiment, the variant comprises an alteration in thepositions corresponding to: 17+20, 17+51, 17+53, 17+55, 17+56, 17+60,17+63, 17+79, 17+87, 17+192, 17+302, 7+387, 17+388, 17+390, 17+403,17+408, 17+410, 17+416, 17+448, 17+451, 17+471, 17+472, 17+507, 17+512,17+515, 17+538, 17+598, 17+602, 17+605, 17+609, 17+676, 17+694, 17+698,17+699, 17+711, 17+754, 17+760, 17+781, 17+786, 17+797, 17+834, 17+835,20+51, 20+53, 20+55, 20+56, 20+60, 20+63, 20+79, 20+87, 20+192, 20+302,20+387, 20+388, 20+390, 20+403, 20+408, 20+410, 20+416, 20+448, 20+451,20+471, 20+472, 20+507, 20+512, 20+515, 20+538, 20+598, 20+602, 20+605,20+609, 20+676, 20+694, 20+698, 20+699, 20+711, 20+754, 20+760, 20+781,20+786, 20+797, 20+834, 20+835, 51+53, 51+55, 51+56, 51+60, 51+63,51+79, 51+87, 51+192, 51+302, 51+387, 51+388, 51+390, 51+403, 51+408,51+410, 51+416, 51+448, 51+451, 51+471, 51+472, 51+507, 51+512, 51+515,51+538, 51+598, 51+602, 51+605, 51+609, 51+676, 51+694, 51+698, 51+699,51+711, 51+754, 51+760, 51+781, 51+786, 51+797, 51+834, 51+835, 53+55,53+56, 53+60, 53+63, 53+79, 53+87, 53+192, 53+302, 53+387, 53+388,53+390, 53+403, 53+408, 53+410, 53+416, 53+448, 53+451, 53+471, 53+472,53+507, 53+512, 53+515, 53+538, 53+598, 53+602, 53+605, 53+609, 3+676,53+694, 53+698, 53+699, 53+711, 53+754, 53+760, 53+781, 53+786, 53+797,53+834, 53+835, 55+56, 55+60, 55+63, 55+79, 55+87, 55+192, 55+302,55+387, 55+388, 55+390, 55+403, 55+408, 55+410, 55+416, 55+448, 55+451,55+471, 55+472, 55+507, 55+512, 55+515, 55+538, 55+598, 55+602, 55+605,55+609, 55+676, 55+694, 55+698, 55+699, 55+711, 55+754, 55+760, 55+781,55+786, 55+797, 55+834, 55+835, 56+60, 56+63, 56+79, 56+87, 56+192,56+302, 56+387, 56+388, 56+390, 56+403, 56+408, 56+410, 56+416, 56+448,56+451, 56+471, 56+472, 56+507, 56+512, 56+515, 56+538, 56+598, 56+602,56+605, 56+609, 56+676, 56+694, 56+698, 56+699, 56+711, 56+754, 56+760,56+781, 56+786, 56+797, 56+834, 56+835, 60+63, 60+79, 60+87, 60+192,60+302, 60+387, 60+388, 60+390, 60+403, 60+408, 60+410, 60+416, 60+448,60+451, 60+471, 60+472, 60+507, 60+512, 60+515, 60+538, 60+598, 60+602,60+605, 60+609, 60+676, 60+694, 60+698, 60+699, 60+711, 60+754, 60+760,60+781, 60+786, 60+797, 60+834, 60+835, 63+79, 63+87, 63+192, 63+302,63+387, 63+388, 63+390, 63+403, 63+408, 63+410, 63+416, 63+448, 63+451,63+471, 63+472, 63+507, 63+512, 63+515, 63+538, 63+598, 63+602, 63+605,63+609, 63+676, 63+694, 63+698, 63+699, 63+711, 63+754, 63+760, 63+781,63+786, 63+797, 63+834, 63+835, 79+87, 79+192, 79+302, 79+387, 79+388,79+390, 79+403, 79+408, 79+410, 79+416, 79+448, 79+451, 79+471, 79+472,79+507, 79+512, 79+515, 79+538, 79+598, 79+602, 79+605, 79+609, 79+676,79+694, 79+698, 79+699, 79+711, 79+754, 79+760, 79+781, 79+786, 79+797,79+834, 79+835, 87+192, 87+302, 87+387, 87+388, 87+390, 87+403, 87+408,87+410, 87+416, 87+448, 87+451, 87+471, 87+472, 87+507, 87+512, 87+515,87+538, 87+598, 87+602, 87+605, 87+609, 87+676, 87+694, 87+698, 87+699,87+711, 87+754, 87+760, 87+781, 87+786, 87+797, 87+834, 87+835, 192+302,192+387, 192+388, 192+390, 192+403, 192+408, 192+410, 192+416, 192+448,192+451, 192+471, 192+472, 192+507, 192+512, 192+515, 192+538, 192+598,192+602, 192+605, 192+609, 192+676, 192+694, 192+698, 192+699, 192+711,192+754, 192+760, 192+781, 192+786, 192+797, 192+834, 192+835, 302+387,302+388, 302+390, 302+403, 302+408, 302+410, 302+416, 302+448, 302+451,302+471, 302+472, 302+507, 302+512, 302+515, 302+538, 302+598, 302+602,302+605, 302+609, 302+676, 302+694, 302+698, 302+699, 302+711, 302+754,302+760, 302+781, 302+786, 302+797, 302+834, 302+835, 387+388, 387+390,387+403, 387+408, 387+410, 387+416, 387+448, 387+451, 387+471, 387+472,387+507, 387+512, 387+515, 387+538, 387+598, 387+602, 387+605, 87+609,387+676, 387+694, 387+698, 387+699, 387+711, 387+754, 387+760, 387+781,387+786, 387+797, 387+834, 387+835, 388+390, 388+403, 388+408, 388+410,388+416, 388+448, 388+451, 388+471, 388+472, 388+507, 388+512, 388+515,388+538, 388+598, 388+602, 388+605, 388+609, 388+676, 388+694, 388+698,388+699, 388+711, 388+754, 388+760, 388+781, 388+786, 388+797, 388+834,388+835, 390+403, 390+408, 390+410, 390+416, 390+448, 390+451, 390+471,390+472, 390+507, 390+512, 390+515, 390+538, 390+598, 390+602, 390+605,390+609, 390+676, 390+694, 390+698, 390+699, 390+711, 390+754, 390+760,390+781, 390+786, 390+797, 390+834, 390+835, 403+408, 403+410, 403+416,403+448, 403+451, 403+471, 403+472, 403+507, 403+512, 403+515, 403+538,403+598, 403+602, 403+605, 403+609, 403+676, 403+694, 403+698, 403+699,403+711, 403+754, 403+760, 403+781, 403+786, 403+797, 403+834, 403+835,408+410, 408+416, 408+448, 408+451, 408+471, 408+472, 408+507, 408+512,408+515, 408+538, 408+598, 408+602, 408+605, 408+609, 408+676, 408+694,408+698, 408+699, 408+711, 408+754, 408+760, 408+781, 408+786, 408+797,408+834, 408+835, 410+416, 410+448, 410+451, 410+471, 410+472, 410+507,410+512, 410+515, 410+538, 410+598, 410+602, 410+605, 410+609, 410+676,410+694, 410+698, 410+699, 410+711, 410+754, 410+760, 410+781, 410+786,410+797, 410+834, 410+835, 416+448, 416+451, 416+471, 416+472, 416+507,416+512, 416+515, 416+538, 416+598, 416+602, 416+605, 416+609, 416+676,416+694, 416+698, 416+699, 416+711, 416+754, 416+760, 416+781, 416+786,416+797, 416+834, 416+835, 448+451, 448+471, 448+472, 448+507, 448+512,448+515, 448+538, 448+598, 448+602, 448+605, 448+609, 448+676, 448+694,448+698, 448+699, 448+711, 448+754, 448+760, 448+781, 448+786, 448+797,448+834, 448+835, 451+471, 451+472, 451+507, 451+512, 451+515, 451+538,451+598, 451+602, 451+605, 451+609, 451+676, 451+694, 451+698, 451+699,451+711, 451+754, 451+760, 451+781, 451+786, 451+797, 451+834, 451+835,471+472, 471+507, 471+512, 471+515, 471+538, 471+598, 471+602, 471+605,471+609, 471+676, 471+694, 471+698, 471+699, 471+711, 471+754, 471+760,471+781, 471+786, 471+797, 471+834, 471+835, 472+507, 472+512, 472+515,472+538, 472+598, 472+602, 472+605, 472+609, 472+676, 472+694, 472+698,472+699, 472+711, 472+754, 472+760, 472+781, 472+786, 472+797, 472+834,472+835, 507+512, 507+515, 507+538, 507+598, 507+602, 507+605, 507+609,507+676, 507+694, 507+698, 507+699, 507+711, 507+754, 507+760, 507+781,507+786, 507+797, 507+834, 507+835, 512+515, 512+538, 512+598, 512+602,512+605, 512+609, 512+676, 512+694, 512+698, 512+699, 512+711, 512+754,512+760, 512+781, 512+786, 512+797, 512+834, 512+835, 515+538, 515+598,515+602, 515+605, 515+609, 515+676, 515+694, 515+698, 515+699, 515+711,515+754, 515+760, 515+781, 515+786, 515+797, 515+834, 515+835, 538+598,538+602, 538+605, 538+609, 538+676, 538+694, 538+698, 538+699, 538+711,538+754, 538+760, 538+781, 538+786, 538+797, 538+834, 538+835, 598+602,598+605, 598+609, 598+676, 598+694, 598+698, 598+699, 598+711, 598+754,598+760, 598+781, 598+786, 598+797, 598+834, 598+835, 602+605, 602+609,602+676, 602+694, 602+698, 602+699, 602+711, 602+754, 602+760, 602+781,602+786, 602+797, 602+834, 602+835, 605+609, 605+676, 605+694, 605+698,605+699, 605+711, 605+754, 605+760, 605+781, 605+786, 605+797, 605+834,605+835, 609+676, 609+694, 609+698, 609+699, 609+711, 609+754, 609+760,609+781, 609+786, 609+797, 609+834, 609+835, 676+694, 676+698, 676+699,676+711, 676+754, 676+760, 676+781, 676+786, 676+797, 676+834, 676+835,694+698, 694+699, 694+711, 694+754, 694+760, 694+781, 694+786, 694+797,694+834, 694+835, 698+699, 698+711, 698+754, 698+760, 698+781, 698+786,698+797, 698+834, 698+835, 699+711, 699+754, 699+760, 699+781, 699+786,699+797, 699+834, 699+835, 711+754, 711+760, 711+781, 711+786, 711+797,711+834, 711+835, 754+760, 754+781, 754+786, 754+797, 754+834, 754+835760+781, 760+786, 760+797, 760+834, 760+835, 781+786, 781+797, 781+834,781+835,786+797,786+834,786+835,797+834,797+835,834+835, 17+20+51,17+20+53, 17+20+55,17+20+56,17+20+60,17+20+63,17+20+79,17+20+87,17+20+192,17+20+302,17+20+387,17+20+388,17+20+390, 17+20+403, 17+20+408, 17+20+410, 17+20+416, 17+20+448,17+20+451, 17+20+471, 7+20+472, 17+20+507, 17+20+512, 17+20+515,17+20+538, 17+20+598, 17+20+602, 17+20+605, 17+20+609, 17+20+676,17+20+694, 17+20+698, 17+20+699, 17+20+711, 17+20+754, 17+20+760,17+20+781, 17+20+786, 17+20+797, 17+20+834, 17+20+835, 17+51+53,17+51+55, 17+51+56, 17+51+60, 17+51+63, 17+51+79, 17+51+87, 17+51+192,17+51+302, 17+51+387, 17+51+388, 17+51+390, 17+51+403, 17+51+408,17+51+410, 17+51+416, 17+51+448, 17+51+451, 17+51+471, 17+51+472,17+51+507, 17+51+512, 17+51+515, 17+51+538, 17+51+598, 17+51+602,17+51+605, 17+51+609, 17+51+676, 17+51+694, 17+51+698, 17+51+699,17+51+711, 17+51+754, 17+51+760, 17+51+781, 17+51+786, 17+51+797,17+51+834, 17+51+835, 17+53+55, 17+53+56, 17+53+60, 17+53+63, 17+53+79,17+53+87, 17+53+192, 17+53+302, 17+53+387, 17+53+388, 17+53+390,17+53+403, 17+53+408, 17+53+410, 17+53+416, 17+53+448, 17+53+451,17+53+471, 17+53+472, 17+53+507, 17+53+512, 17+53+515, 17+53+538,17+53+598, 17+53+602, 17+53+605, 17+53+609, 17+53+676, 17+53+694,17+53+698, 17+53+699, 17+53+711, 17+53+754, 17+53+760, 17+53+781,17+53+786,17+53+797,17+53+834,17+53+835,17+55+56,17+55+60,17+55+63,17+55+79,17+55+87,17+55+192, 17+55+302, 17+55+387, 17+55+388, 17+55+390, 17+55+403,17+55+408, 17+55+410, 17+55+416, 17+55+448, 17+55+451, 17+55+471,17+55+472, 17+55+507, 17+55+512, 17+55+515, 17+55+538, 17+55+598,17+55+602, 17+55+605, 17+55+609, 17+55+676, 17+55+694, 17+55+698,17+55+699, 17+55+711, 17+55+754, 17+55+760, 17+55+781, 17+55+786,17+55+797, 17+55+834, 17+55+835, 17+56+60, 17+56+63, 17+56+79, 17+56+87,17+56+192, 17+56+302, 17+56+387, 17+56+388, 17+56+390, 17+56+403,17+56+408, 17+56+410, 17+56+416, 17+56+448, 17+56+451, 17+56+471,17+56+472, 17+56+507, 17+56+512, 17+56+515, 17+56+538, 17+56+598,17+56+602, 17+56+605, 17+56+609, 17+56+676, 17+56+694, 17+56+698,17+56+699, 17+56+711, 17+56+754, 17+56+760, 17+56+781, 17+56+786,17+56+797, 17+56+834, 17+56+835, 17+60+63, 17+60+79, 17+60+87,17+60+192, 17+60+302, 17+60+387, 17+60+388, 17+60+390, 17+60+403,17+60+408, 17+60+410, 17+60+416, 17+60+448, 17+60+451, 17+60+471,17+60+472, 17+60+507, 17+60+512, 17+60+515, 17+60+538, 17+60+598,17+60+602, 17+60+605, 17+60+609, 17+60+676, 17+60+694, 17+60+698,17+60+699, 17+60+711, 17+60+754, 17+60+760, 17+60+781, 17+60+786,17+60+797, 17+60+834, 17+60+835, 17+63+79, 17+63+87, 17+63+192,17+63+302, 17+63+387, 17+63+388, 17+63+390, 17+63+403, 17+63+408,17+63+410, 17+63+416, 17+63+448, 17+63+451, 17+63+471, 17+63+472,17+63+507, 17+63+512, 17+63+515, 17+63+538, 17+63+598, 17+63+602,17+63+605, 17+63+609, 17+63+676, 17+63+694, 17+63+698, 17+63+699,17+63+711, 17+63+754, 17+63+760, 17+63+781, 17+63+786, 17+63+797,17+63+834, 17+63+835, 17+79+87, 17+79+192, 17+79+302, 17+79+387,17+79+388, 17+79+390, 17+79+403, 17+79+408, 17+79+410, 17+79+416,17+79+448, 17+79+451, 17+79+471, 17+79+472, 17+79+507, 17+79+512,17+79+515, 17+79+538, 17+79+598, 17+79+602, 17+79+605, 17+79+609,17+79+676, 17+79+694, 17+79+698, 17+79+699, 17+79+711, 17+79+754,17+79+760, 17+79+781, 17+79+786, 17+79+797, 17+79+834, 17+79+835,17+87+192, 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711+797+834, 711+797+835, 711+834+835, 754+760+781,754+760+786, 754+760+797, 754+760+834, 754+760+835, 754+781+786,754+781+797, 754+781+834, 754+781+835, 754+786+797, 754+786+834,754+786+835, 754+797+834, 754+797+835, 754+834+835, 760+781+786,60+781+797, 760+781+834, 760+781+835, 760+786+797, 760+786+834,760+786+835, 760+797+834, 760+797+835 760+834+835, 781+786+797,781+786+834, 781+786+835, 781+797+834, 781+797+835, 781+834+835,786+797+834, 786+797+835, 786+834+835, and 797+834+835 of SEQ ID NO: 2.

In another aspect, a variant comprises an alteration at each position(or at least four positions) corresponding to positions 17, 20, 51, 53,55, 56, 60, 63, 79, 87, 186, 192, 302, 311, 313, 387, 388, 390, 403,408, 410, 416, 448, 451, 471, 472, 476, 489, 507, 512, 515, 538, 598,599, 602, 603, 605, 609, 676, 688, 690, 694, 697, 698, 699, 711, 719,754, 756, 760, 781, 786, 797, 833, 834, 835 and 1048 of SEQ ID NO: 2,wherein numbering is according to SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 17. In another aspect, the aminoacid at a position corresponding to position 17 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution 517A of the mature polypeptide of SEQ IDNO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 20. In another aspect, the aminoacid at a position corresponding to position 20 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution F20P, F20N, F20G, or F20Y, of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 51. In another aspect, the aminoacid at a position corresponding to position 51 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution K51Q or K51H, of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 53. In another aspect, the aminoacid at a position corresponding to position 53 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution E53P or E53Q, of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 55. In another aspect, the aminoacid at a position corresponding to position 55 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution Y55M of the mature polypeptide of SEQ IDNO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 56. In another aspect, the aminoacid at a position corresponding to position 56 is substituted with Ala,Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution V56M of the mature polypeptide of SEQ IDNO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 60. In another aspect, the aminoacid at a position corresponding to position 60 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution Y60F of the mature polypeptide of SEQ IDNO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 63. In another aspect, the aminoacid at a position corresponding to position 63 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution S63F of the mature polypeptide of SEQ IDNO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 79. In another aspect, the aminoacid at a position corresponding to position 79 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution S79W of the mature polypeptide of SEQ IDNO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 87. In another aspect, the aminoacid at a position corresponding to position 87 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution T87R of the mature polypeptide of SEQ IDNO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 186. In another aspect, the aminoacid at a position corresponding to position 186 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A186P of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 192. In another aspect, the aminoacid at a position corresponding to position 192 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K192N of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 302. In another aspect, the aminoacid at a position corresponding to position 302 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution 1302D, 1302V, 1302M or 1302H,of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 311. In another aspect, the aminoacid at a position corresponding to position 311 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution H311N, of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 313. In another aspect, the aminoacid at a position corresponding to position 302 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S313D of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 387. In another aspect, the aminoacid at a position corresponding to position 387 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution 1387T of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 388. In another aspect, the aminoacid at a position corresponding to position 388 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K388R of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 390. In another aspect, the aminoacid at a position corresponding to position 390 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K390Q of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 403. In another aspect, the aminoacid at a position corresponding to position 403 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution 1403Y of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 408. In another aspect, the aminoacid at a position corresponding to position 408 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the alteration E408D, E408S, E408P, E408A,E408G, E408N, of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 410. In another aspect, the aminoacid at a position corresponding to position 410 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution P410G of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 416. In another aspect, the aminoacid at a position corresponding to position 416 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution Q416S or Q416D, of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 448. In another aspect, the aminoacid at a position corresponding to position 448 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A448E, A448S or A448W, of themature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 451. In another aspect, the aminoacid at a position corresponding to position 451 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K451Q and K451S, of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 471. In another aspect, the aminoacid at a position corresponding to position 471 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution G471S of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 472. In another aspect, the aminoacid at a position corresponding to position 472 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S472Y of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 476. In another aspect, the aminoacid at a position corresponding to position 476 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution D476R of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 489. In another aspect, the aminoacid at a position corresponding to position 489 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution Q489P of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 507. In another aspect, the aminoacid at a position corresponding to position 507 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K507R of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 512. In another aspect, the aminoacid at a position corresponding to position 512 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K512P of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 515. In another aspect, the aminoacid at a position corresponding to position 515 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S515V of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 538. In another aspect, the aminoacid at a position corresponding to position 538 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S538C of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 598. In another aspect, the aminoacid at a position corresponding to position 598 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S598Q of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 599. In another aspect, the aminoacid at a position corresponding to position 599 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A599S of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 602. In another aspect, the aminoacid at a position corresponding to position 602 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution 1602T or 1602D, of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 603. In another aspect, the aminoacid at a position corresponding to position 603 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution V603P of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 605. In another aspect, the aminoacid at a position corresponding to position 605 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution 5605T of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 609. In another aspect, the aminoacid at a position corresponding to position 609 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution G609E of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 676. In another aspect, the aminoacid at a position corresponding to position 676 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution D676H of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 688. In another aspect, the aminoacid at a position corresponding to position 688 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A688G of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 690. In another aspect, the aminoacid at a position corresponding to position 690 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution Y690F of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 694. In another aspect, the aminoacid at a position corresponding to position 694 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T694A of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 697. In another aspect, the aminoacid at a position corresponding to position 697 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T697G of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 698. In another aspect, the aminoacid at a position corresponding to position 698 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution R698W of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 699. In another aspect, the aminoacid at a position corresponding to position 699 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T699A of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 711. In another aspect, the aminoacid at a position corresponding to position 711 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T711V or T711T, of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 719. In another aspect, the aminoacid at a position corresponding to position 719 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution W719R of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 754. In another aspect, the aminoacid at a position corresponding to position 754 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution K754R of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 756. In another aspect, the aminoacid at a position corresponding to position 756 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution V756H or V756Y of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 760. In another aspect, the aminoacid at a position corresponding to position 760 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S760G of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 781. In another aspect, the aminoacid at a position corresponding to position 781 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T781M of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 786. In another aspect, the aminoacid at a position corresponding to position 786 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution N786K of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 797. In another aspect, the aminoacid at a position corresponding to position 797 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution T797S of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 824. In another aspect, the aminoacid at a position corresponding to position 824 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A824D of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 833. In another aspect, the aminoacid at a position corresponding to position 833 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution N833D of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 834. In another aspect, the aminoacid at a position corresponding to position 834 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution Q834E of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 835. In another aspect, the aminoacid at a position corresponding to position 835 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution S835D of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position 1048. In another aspect, the aminoacid at a position corresponding to position 1048 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution F1048W of the maturepolypeptide of SEQ ID NO: 2.

In one embodiment, the endoglucanase variant comprised in the detergentcompositions may further comprise an alteration at one or more positionsselected from the group consisting of alterations in positions:

216, 283, 346, 559, 564, 579, 636, 638, 651, 824, 848, 869, 880, 881,883, 887, 892, 905, 906, 912, 921, 928, 934, 937, 948, 956, 999 and1037, or corresponding to those positions.

More specifically the endoglucanase variant comprised in the detergentcompositions may further comprise one or more alterations at one or morepositions selected from the group consisting of: N216D, N216Q, A283D,A346D, A559P, A559N, A564E, Y579W, S636K, S636N, F638N, A651P, A824D,N848D, A869V, R880K, V881T, V881Q, T883R, T887K, T892P, N905D, F906A,A912V, K921R, S928D, Y934G, A937E, K948R, Q956Y, T999R, and A1037E, orcorresponding to those positions.

In another aspect, the variant comprises or consists of an alteration ata position corresponding to position selected from the group consistingof alterations in positions: 17, 20, 51, 53, 55, 56, 60, 63, 79, 87,186, 192, 302, 311, 313, 387, 388, 390, 403, 408, 410, 416, 448, 451,471, 472, 476, 489, 507, 512, 515, 538, 598, 599, 602, 603, 605, 609,676, 688, 690, 694, 697, 698, 699, 711, 719, 754, 756, 760, 781, 786,797, 834, 833, 835 and 1048 of SEQ ID NO: 2, wherein numbering isaccording to SEQ ID NO: 2.

In another aspect, the amino acid at a position corresponding to any ofpositions as described above is substituted with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In another aspect, the variant comprises or consists of thesubstitution selected from the group consisting of: 517A, F20P, F20N,F20G, F20Y, K51Q, K51H, E53P, E53G, Y55M, V56M, Y60F, S63F, T87R, A186P,K192N, 1302D, 1302H, 1302V, 1302M, H311N, S313D, I387T, K388R, K390Q,1403Y, E408D, E408S, E408P, E408A, E408G, E408N, P410G, Q416S, Q416D,A448E, A448W, A448S, K451S, G471S, S472Y, D476R, Q489P, K507R, K512P,S515V, S538C, Y579W, S598Q, A599S, 1602T, 1602D, V603P, 5605T, G609E,D676H, A688G, Y690F, T694A, T697G, R698W, T699A, T711V, T711Y, W719R,K754R, V756H, V756Y, 5760G, T781M, N786K, T797S, A824D, N833D, Q834E,S835D and F1048W, wherein numbering is according to SEQ ID NO: 2.

In a particular embodiment, a detergent composition may include anendoglucanase variant selected from the group consisting of theendoglucananase variants set forth in Table 2 herein.

In a particular embodiment, a detergent composition may include anendoglucananase variant selected from the group consisting of theendoglucananase variants set forth in Table 3 herein.

In a particular embodiment, a detergent composition may include anendoglucananase variant selected from the group consisting of theendoglucananase variants set forth in Table 4 herein.

In a particular embodiment, a detergent composition may include anendoglucananase variant selected from the group consisting of theendoglucananase variants set forth in Table 5 herein.

In a particular embodiment, a detergent composition may include anendoglucananase variant selected from the group consisting of theendoglucananase variants set forth in Table 6 herein.

The variants may further comprise one or more additional alterations atone or more (e.g., several) other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andaspara-gine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryp-tophan and tyrosine), andsmall amino acids (glycine, alanine, serine, threonine and methionine).Amino acid substitutions that do not generally alter specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for xanthan lyase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron dif-fraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

In one embodiment, a detergent composition may include an endoglucanasevariant, having the total number of alterations compared to SEQ ID NO: 2between 1 and 20, e.g., between 1 and 10 or between 1 and 5, such as 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.

In one embodiment, a detergent composition may include an endoglucanasevariant, wherein an activity on xanthan gum pre-treated with xanthanlyase is a xanthan degrading activity, said xanthan degrading activityis endoglucanase EC 3.2.1.4 activity.

In an embodiment, the variant has an improved stability in a detergentcomposition compared to a parent enzyme (e.g., SEQ ID NO: 2).

In one embodiment, a detergent composition may include an endoglucanasevariant, wherein said variant has an improved stability in a detergentcomposition compared to a parent endoglucanase (e.g., with SEQ ID NO:2).

In one embodiment, a detergent composition may include an endoglucanasevariant, wherein said variant has a half-life improvement factor(HIF)≥1.0; said variant has a half-life improvement factor (HIF)>1.0relative to a parent endoglucanase, e.g. an endoglucanase of SEQ ID NO:2. A way of calculating said half-life improvement factor (HIF) isdescribed in example 3 herein. Accordingly, residual activity (RA) canbe calculated using the following formula:

${{RA}(\%)} = {\frac{{Abs}({Stress})}{{Abs}({Ref})} \times 100\%}$

wherein Abs(Stress) is the absorbance at 405 nm of the sample in thestress microtiter plate (MTP) (e.g. incubated at 25° C. over-night orany other combination of temperature and time) after subtractingrelevant background absorbance contributions, Abs(Ref) is the absorbanceat 405 nm of the sample in the reference MTP (e.g. incubated at 5° C.over-night) after subtracting relevant background absorbancecontributions, wherein half-lives for the degradation of each variantand parent endoglucanase (e.g. at 25° C.) are calculated using thefollowing formula:

${T\; 1\text{/}2} = {- \frac{{\ln (2)} \times T}{\ln \left( \frac{{Abs}({Stress})}{{Abs}({Ref})} \right)}}$

wherein T is the incubation time for both the stress and reference MTP,wherein half-life-improvement factors (HIFs) are calculated using thefollowing formula:

${HIF} = \frac{{T\; 1\text{/}2},{variant}}{{T\; 1\text{/}2},{wt}}$

e.g. wherein T1/2 wt (or wild type) is the T1/2 of the mature parentendoglucanase with SEQ ID NO: 2.

In one embodiment, a detergent composition may include an endoglucanasevariant, wherein a half-life improvement factor (HIF) is determinedafter incubation of said endoglucanase variant in a detergentcomposition at 25° C. for a time period from about 17 to about 20 hours.

Parent

The parent endoglucanase may be (a) a polypeptide having at least 60%sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, or (ii) the full-length complement of (i); or (c) apolypeptide encoded by a polynucleotide having at least 60% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which havexanthan lyase activity. In one aspect, the amino acid sequence of theparent differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10, from the mature polypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent is a fragment of the mature polypeptide of SEQ ID NO: 2containing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or95% of the number of amino acids of SEQ ID NO: 2. In another embodiment,the parent is an allelic variant of the mature polypeptide of SEQ ID NO:2.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,New York).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. In a non-limiting embodiment, the nucleic acid probe is at least100 nucleotides in length, e.g., at least 200 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 600 nucleotides, at least 700 nucleotides, at least 800nucleotides, or at least 900 nucleotides in length. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

Hybridization indicates that the polynucleotide hybridizes to a labelednucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the maturepolypeptide coding sequence of SEQ ID NO: 1; (iii) the full-lengthcom-element thereof; or (iv) a subsequence thereof; under very low tovery high stringency conditions. Molecules to which the nucleic acidprobe hybridizes under these conditions can be detected using, forexample, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe is apolynucleotide that encodes the polypeptide of SEQ ID NO: 2; the maturepolypeptide thereof; or a fragment thereof. In another aspect, thenucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide. A fusion polypeptide is produced byfusing a polynucleotide encoding another polypeptide to apolynucleotide. Techniques for producing fusion polypeptides are knownin the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusionpolypeptide is under control of the same promoter(s) and terminator.Fusion polypeptides may also be constructed using intein technology inwhich fusion polypeptides are created post-translationally (Cooper etal., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266:776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes herein, the term “obtained from” as used herein in connectionwith a given source shall mean that the parent encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, theparent is secreted extracellularly.

The parent may be a bacterial enzyme. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces enzyme, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma enzyme.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis enzyme.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. zooepidemicus enzyme.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans enzyme.

The parent may be a fungal enzyme. For example, the parent may be ayeast enzyme such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia enzyme; or a filamentous fungal enzymesuch as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria enzyme.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis enzyme.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride enzyme.

In another aspect, the parent is a Paenibacillus sp. xanthan lyase,e.g., the xanthan lyase of SEQ ID NO: 2.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

Methods for obtaining a variant having endoglucanase activity, maycomprise: (a) introducing into a parent endoglucanase an alteration atone or more (e.g., several) positions corresponding to positions 17, 20,51, 53, 55, 56, 60, 63, 79, 87, 186, 192, 302, 311, 313, 387, 388, 390,403, 408, 410, 416, 448, 451, 471, 472, 476, 489, 507, 512, 515, 538,598, 599, 602, 603, 605, 609, 676, 688, 690, 694, 697, 698, 699, 711,719, 754, 756, 760, 781, 786, 797, 833, 834, 835 and 1048 of SEQ ID NO:2 of the mature polypeptide of SEQ ID NO: 2, wherein the variant hasendoglucanase activity; and (b) recovering the variant.

In an embodiment, the variant to be obtained may further comprise analteration at one or more positions corresponding to positions selectedfrom the group consisting of alterations in positions: 216, 283, 346,559, 564, 579, 636, 638, 651, 824, 848, 869, 880, 881, 883, 887, 892,905, 906, 912, 921, 928, 934, 937, 948, 956, 999 and 1037 of SEQ ID NO:2 of the mature polypeptide of SEQ ID NO: 2, wherein the variant hasendoglucanase activity; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used. There are manycommercial kits available that can be used to prepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microflu-idicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Embodiments

In one embodiment, to a detergent composition may include at least one(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) endoglucanase variant.

In one embodiment, a detergent composition may include at least one(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) endoglucanase variant, furthercomprising one or more detergent components.

In one embodiment, a detergent composition may include at least one(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) endoglucanase variant, furthercomprising one or more additional enzymes selected from the groupconsisting of: xanthan lyases, proteases, amylases, lichenases, lipases,cutinases, cellulases, endoglucanases, xyloglucanases, pectinases,pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalasesand mannanases, or any mixture thereof.

In one embodiment, a detergent composition may include at least one(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) endoglucanase variant, furthercomprising one or more detergent components and one or more additionalenzymes selected from the group consisting of: xanthan lyases,proteases, amylases, lichenases, lipases, cutinases, cellulases,endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidases, haloperoxygenases, catalases and mannanases, or any mixturethereof.

In one embodiment, a detergent composition may include at least one(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) endoglucanase variant, furthercomprising one or more detergent components, wherein said detergentcomposition is in form of a bar, a homogenous tablet, a tablet havingtwo or more layers, a pouch having one or more compartments, a regularor compact powder, a granule, a paste, a gel, or a regular, compact orconcentrated liquid.

In one embodiment, a detergent composition, may be used for degradingxanthan gum and use in a cleaning process, such as laundry or hardsurface cleaning such as dish wash.

In one embodiment, a detergent composition may have an enzyme detergencybenefit

In one embodiment, a method for degrading xanthan gum may include:applying a detergent composition to a xanthan gum.

In one embodiment, a method for degrading xanthan gum may include:applying a detergent composition to a xanthan gum, wherein said xanthangum is on the surface of a textile or hard surface, such as dish wash.

Polynucleotides

Also disclosed herein are isolated polynucleotides encoding a variant.Accordingly, in one aspect, the present disclosure relates to isolatedpolynucleotides encoding a variant comprising an alteration at one ormore positions in a region selected from the group consisting of: i)region 10 corresponding to amino acids 1 to 94 of SEQ ID NO: 2, ii)region 11 corresponding to amino acids 106 to 114 of SEQ ID NO: 2, iii)region 12 corresponding to amino acids 139 to 209 of SEQ ID NO: 2, iv)region 13 corresponding to amino acids 252 to 266 of SEQ ID NO: 2, v)region 14 corresponding to amino acids 302 to 338 of SEQ ID NO: 2, vi)region 15 corresponding to amino acids 362 to 546 of SEQ ID NO: 2, vii)region 16 corresponding to amino acids 596 to 611 of SEQ ID NO: 2, viii)region 17 corresponding to amino acids 661 to 805 of SEQ ID NO: 2, ix)region 18 corresponding to amino acids 829 to 838 of SEQ ID NO: 2, andx) region 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2,wherein said variant has at least 60% and less than 100% sequenceidentity to SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleo-tide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned in a strain of Bacillus subtilis or E.coli, or a related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of thepolynucleotide.

Modification of a polynucleotide encoding a polypeptide may be necessaryfor synthesizing polypeptides substantially similar to the polypeptide.The term “substantially similar” to the polypeptide refers tonon-naturally occurring forms of the polypeptide. These polypeptides maydiffer in some engineered way from the polypeptide isolated from itsnative source, e.g., variants that differ in specific activity,thermostability, pH optimum, or the like. The variants may beconstructed on the basis of the polynucleotide presented as the maturepolypeptide coding sequence of SEQ ID NO: 1, e.g., a subsequencethereof, and/or by introduction of nucleotide substitutions that do notresult in a change in the amino acid sequence of the polypeptide, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, Ford et al., (1991),‘Protein Expression and Purification’, 2: 95-107.

Nucleic Acid Constructs

Also disclosed herein are nucleic acid constructs comprising apolynucleotide encoding a variant as described herein operably linked toone or more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Accordingly, disclosed are nucleic acid constructscomprising a polynucleotide encoding a variant comprising an alterationat one or more positions in a region selected from the group consistingof: i) region 10 corresponding to amino acids 1 to 94 of SEQ ID NO: 2,ii) region 11 corresponding to amino acids 106 to 114 of SEQ ID NO: 2,iii) region 12 corresponding to amino acids 139 to 209 of SEQ ID NO: 2,iv) region 13 corresponding to amino acids 252 to 266 of SEQ ID NO: 2,v) region 14 corresponding to amino acids 302 to 338 of SEQ ID NO: 2,vi) region 15 corresponding to amino acids 362 to 546 of SEQ ID NO: 2,vii) region 16 corresponding to amino acids 596 to 611 of SEQ ID NO: 2,viii) region 17 corresponding to amino acids 661 to 805 of SEQ ID NO: 2,ix) region 18 corresponding to amino acids 829 to 838 of SEQ ID NO: 2,and x) region 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO:2, wherein said variant has at least 60% and less than 100% sequenceidentity to SEQ ID NO: 2, operably linked to one or more controlsequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary depend-ingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide. The promoter contains transcriptional control sequencesthat mediate the expression of the polypeptide. The promoter may be anypolynucleotide that shows transcriptional activity in the host cellincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs in a bacterial host cell are the promotersobtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ),Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformispenicillinase gene (penP), Bacillus stearothermophilus maltogenicamylase gene (amyM), Bacillus subtilis levansucrase gene (sacB),Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIAgene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American 242: 74-94; and in Sambrook et al., 1989, supra.Examples of tandem promoters are disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus nidulans acetamidase,Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkalineprotease, Aspergillus oryzae triose phosphate isomerase, Fusariumoxysporum trypsin-like protease (WO 96/00787), Fusarium venenatumamyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900),Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase,Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydro-genase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI),Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomycescerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeasthost cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused.

Non-limiting terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Non-limiting terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Non-limiting terminators for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceralde-hyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Non-limiting leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Non-limiting polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (npr7),Myceliophthora thermophile laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

Disclosed herein are recombinant expression vectors comprising apolynucleotide encoding a variant as described herein, a promoter, andtranscriptional and translational stop signals. Accordingly, the presentdisclosure relates to recombinant expression vectors comprising apolynucleotide encoding a variant comprising an alteration at one ormore positions in a region selected from the group consisting of: i)region 10 corresponding to amino acids 1 to 94 of SEQ ID NO: 2, ii)region 11 corresponding to amino acids 106 to 114 of SEQ ID NO: 2, iii)region 12 corresponding to amino acids 139 to 209 of SEQ ID NO: 2, iv)region 13 corresponding to amino acids 252 to 266 of SEQ ID NO: 2, v)region 14 corresponding to amino acids 302 to 338 of SEQ ID NO: 2, vi)region 15 corresponding to amino acids 362 to 546 of SEQ ID NO: 2, vii)region 16 corresponding to amino acids 596 to 611 of SEQ ID NO: 2, viii)region 17 corresponding to amino acids 661 to 805 of SEQ ID NO: 2, ix)region 18 corresponding to amino acids 829 to 838 of SEQ ID NO: 2, andx) region 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2,wherein said variant has at least 60% and less than 100% sequenceidentity to SEQ ID NO: 2, a promoter, and transcriptional andtranslational stop signals.

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the polypeptide at such sites.Alternatively, the polynucleotide may be expressed by inserting thepolynucleotide or a nucleic acid construct comprising the polynucleotideinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conven-iently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromoso-mal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachro-mosomal element, a minichromosome, or an artificialchromosome. The vector may contain any means for assuringself-replication. Alternatively, the vector may be one that, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids that together contain the total DNA to be introducedinto the genome of the host cell, or a transposon, may be used.

The vector contains one or more selectable markers that permit easyselection of transformed, trans-fected, transduced, or the like cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spec-tinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof Aspergillus nidulans or Aspergillus oryzae amdS andpyrG genes and a Streptomyces hygroscopicus bar gene may be used in anAspergillus cell.

The vector contains an element(s) that permits integration of the vectorinto the host cell's genome or autonomous replication of the vector inthe cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAIV1111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide may be inserted into a host cellto increase production of a polypeptide. An increase in the copy numberof the polynucleotide can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the polynucleotidewhere cells containing amplified copies of the selectable marker gene,and thereby additional copies of the polynucleotide, can be selected forby cultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

Further disclosed are recombinant host cells, comprising apolynucleotide encoding a variant as described herein operably linked toone or more control sequences that direct the production of a variant.Accordingly, in one aspect, the present disclosure relates torecombinant host cells, comprising a polynucleotide encoding a variantcomprising an alteration at one or more positions in a region selectedfrom the group consisting of: i) region 10 corresponding to amino acids1 to 94 of SEQ ID NO: 2, ii) region 11 corresponding to amino acids 106to 114 of SEQ ID NO: 2, iii) region 12 corresponding to amino acids 139to 209 of SEQ ID NO: 2, iv) region 13 corresponding to amino acids 252to 266 of SEQ ID NO: 2, v) region 14 corresponding to amino acids 302 to338 of SEQ ID NO: 2, vi) region 15 corresponding to amino acids 362 to546 of SEQ ID NO: 2, vii) region 16 corresponding to amino acids 596 to611 of SEQ ID NO: 2, viii) region 17 corresponding to amino acids 661 to805 of SEQ ID NO: 2, ix) region 18 corresponding to amino acids 829 to838 of SEQ ID NO: 2, and x) region 19 corresponding to amino acids 1043to 1055 of SEQ ID NO: 2, wherein said variant has at least 60% and lessthan 100% sequence identity to SEQ ID NO: 2, operably linked to one ormore control sequences that direct the production of a variantcomprising an alteration at one or more positions in a selected from thegroup consisting of: i) region 10 corresponding to amino acids 1 to 94of SEQ ID NO: 2, ii) region 11 corresponding to amino acids 106 to 114of SEQ ID NO: 2, iii) region 12 corresponding to amino acids 139 to 209of SEQ ID NO: 2, iv) region 13 corresponding to amino acids 252 to 266of SEQ ID NO: 2, v) region 14 corresponding to amino acids 302 to 338 ofSEQ ID NO: 2, vi) region 15 corresponding to amino acids 362 to 546 ofSEQ ID NO: 2, vii) region 16 corresponding to amino acids 596 to 611 ofSEQ ID NO: 2, viii) region 17 corresponding to amino acids 661 to 805 ofSEQ ID NO: 2, ix) region 18 corresponding to amino acids 829 to 838 ofSEQ ID NO: 2, and x) region 19 corresponding to amino acids 1043 to 1055of SEQ ID NO: 2, wherein said variant has at least 60% and less than100% sequence identity to SEQ ID NO: 2.

A construct or vector comprising a polynucleotide is introduced into ahost cell so that the construct or vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier. The term “host cell” encompasses any progeny of a parent cellthat is not identical to the parent cell due to mutations that occurduring replication. The choice of a host cell will to a large extentdepend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycots, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes).Since the classification of yeast may change in the future, yeast shallbe defined as described in Biology and Activities of Yeast (Skinner,Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by bud-ding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

Also disclosed are methods of producing (e.g., in vitro or ex vivomethods) a variant, comprising: (a) cultivating a host cell underconditions suitable for expression of the variant; and (b) recoveringthe variant. Accordingly, in one aspect, the present disclosure relatesto methods of producing (e.g. in vitro or ex vivo methods) a variantcomprising an alteration at one or more positions in a region selectedfrom the group consisting of: i) region 10 corresponding to amino acids1 to 94 of SEQ ID NO: 2, ii) region 11 corresponding to amino acids 106to 114 of SEQ ID NO: 2, iii) region 12 corresponding to amino acids 139to 209 of SEQ ID NO: 2, iv) region 13 corresponding to amino acids 252to 266 of SEQ ID NO: 2, v) region 14 corresponding to amino acids 302 to338 of SEQ ID NO: 2, vi) region 15 corresponding to amino acids 362 to546 of SEQ ID NO: 2, vii) region 16 corresponding to amino acids 596 to611 of SEQ ID NO: 2, viii) region 17 corresponding to amino acids 661 to805 of SEQ ID NO: 2, ix) region 18 corresponding to amino acids 829 to838 of SEQ ID NO: 2, and x) region 19 corresponding to amino acids 1043to 1055 of SEQ ID NO: 2, wherein said variant has at least 60% and lessthan 100% sequence identity to SEQ ID NO: 2, the method comprisingcultivating a host cell comprising a polynucleotide encoding thevariant, under conditions suitable for expression of the variant; and(b) optionally, recovering the variant.

Further disclosed are methods of producing (e.g., in vitro or ex vivomethods) a variant as described herein, comprising (a) cultivating acell, which in its wild-type form produces the polypeptide, underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide. In an aspect, the cell is a Paenibacilluscell, or a Microbacterium cell.

Also disclosed are methods of producing (e.g., in vitro or ex vivomethods) a variant as described herein, comprising (a) cultivating arecombinant host cell under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid-state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The variant polypeptide may be detected using methods known in the artthat are specific for the polypeptides such as methods for determiningcellulose or xanthan lyase activity. These detection methods include,but are not limited to, use of specific antibodies, formation of anenzyme product, or disappearance of an enzyme substrate. For example, anenzyme assay may be used to determine the activity of the polypeptide.

The variant polypeptide may be recovered using methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant polypeptide may be purified by a variety of procedures knownin the art including, but not limited to, chromatography (e.g., ionexchange, affinity, hydrophobic, chromatofocusing, and size exclu-sion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the variant polypeptide is not recovered, butrather a host cell expressing the polypeptide is used as a source of thevariant polypeptide.

Corn positions

In one certain aspect, the variants have improved stability indetergents compared to a parent enzyme or compared to an endoglucanasehaving the identical amino acid sequence of the variant, but not havingan alteration (e.g., a substitution, deletion or insertion) at one ormore of the specified positions or compared to the endoglucanase withSEQ ID NO: 2, wherein activity and/or stability in detergent is measuredas disclosed in example 3 herein. Thus, in one embodiment, detergentcompositions may include a variant comprising an alteration at one ormore positions in a region selected from the group consisting of: i)region 10 corresponding to amino acids 1 to 94 of SEQ ID NO: 2, ii)region 11 corresponding to amino acids 106 to 114 of SEQ ID NO: 2, iii)region 12 corresponding to amino acids 139 to 209 of SEQ ID NO: 2, iv)region 13 corresponding to amino acids 252 to 266 of SEQ ID NO: 2, v)region 14 corresponding to amino acids 302 to 338 of SEQ ID NO: 2, vi)region 15 corresponding to amino acids 362 to 546 of SEQ ID NO: 2, vii)region 16 corresponding to amino acids 596 to 611 of SEQ ID NO: 2, viii)region 17 corresponding to amino acids 661 to 805 of SEQ ID NO: 2, ix)region 18 corresponding to amino acids 829 to 838 of SEQ ID NO: 2, andx) region 19 corresponding to amino acids 1043 to 1055 of SEQ ID NO: 2,wherein said variant has at least 60% and less than 100% sequenceidentity to SEQ ID NO: 2.

Besides enzymes the detergent compositions may comprise additionalcomponents. The choice of additional components is within the skill ofthe artisan and includes conventional ingredients, including theexemplary non-limiting components set forth below. The choice ofcomponents may include, for fabric care, the consider-ation of the typeof fabric to be cleaned, the type and/or degree of soiling, thetemperature at which cleaning is to take place, and the formulation ofthe detergent product. Although components mentioned below arecate-gorized by general header according to a particular functionality,this is not to be construed as a limitation, as a component may compriseadditional functionalities as will be appreciated by the skilledartisan.

The detergent composition may be suitable for the laundering of textilessuch as e.g. fabrics, cloths or linen, or for cleaning hard surfacessuch as e.g. floors, tables, or dish wash.

Detergent Compositions

In one embodiment, a variant as described herein may be added to adetergent composition in an amount corresponding to 0.0001-200 mg ofenzyme protein, such as 0.0005-100 mg of enzyme protein, such as0.001-30 mg of enzyme protein, alternatively 0.005-8 mg of enzymeprotein, or 0.01-2 mg of enzyme protein per litre of wash liquor.

A composition for use in automatic dishwash (ADW), for example, maycomprise 0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as0.05-5% of enzyme protein by weight of the composition.

A composition for use in laundry granulation or a solid/granular laundrycomposition in general, for example, may comprise 0.0001%-50%, such as0.001%-20%, such as 0.01%-10%, such as 0.05%-5% of enzyme protein byweight of the composition.

A composition for use in laundry liquid, for example, may comprise0.0001%-10%, such as 0.001-7%, such as 0.1%-5% of enzyme protein byweight of the composition.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and thecomposition may be formulated as described in, for example, WO92/19709and WO92/19708.

In certain markets different wash conditions and, as such, differenttypes of detergents are used. This is disclosed in e.g. EP 1 025 240.For example, In Asia (Japan) a low detergent concentration system isused, while the United States uses a medium detergent concentrationsystem, and Europe uses a high detergent concentration system.

A low detergent concentration system includes detergents where less thanabout 800 ppm of detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of detergent components are present in thewash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500-5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Suchdetergent compositions are all embodiments.

A polypeptide may also be incorporated in the detergent formulationsdisclosed in WO97/07202, which is hereby incorporated by reference.

Surfactants

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more non-ionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized.

When included therein the detergent will usually comprise from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually comprise from about 0%to about 10% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylethanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternaryammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, andcombinations thereof.

When included therein the detergent will usually comprise from about0.2% to about 40% by weight of a non-ionic surfactant, for example fromabout 0.5% to about 30%, in particular from about 1% to about 20%, fromabout 3% to about 10%, such as from about 3% to about 5%, or from about8% to about 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propox-ylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), al-kylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanola-mides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glu-camides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein the detergent will usually comprise from about 0%to about 10% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxy-ethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein the detergent will usually comprise from about 0%to about 10% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbeta-ine, sulfobetaine, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds inaqueous solutions (or oppo-sitely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic and ahydrophobic character (so-called amphiphilic properties as known fromsurfactants); however, the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see e.g. review by Hodgdonand Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmicel-lar, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscontaining substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may comprise 0-5% by weight, such as about 0.5 to about5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known inthe art for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate(STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS),sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers,sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodiumethylhexyl sulfate, and combinations thereof.

Builders and Co-Builders

The detergent composition may comprise about 0-65% by weight, such asabout 5% to about 45% of a detergent builder or co-builder, or a mixturethereof. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. The builder and/or co-builder mayparticularly be a chelating agent that forms water-soluble complexeswith Ca and Mg. Any builder and/or co-builder known in the art for usein laundry detergents may be utilized. Non-limiting examples of buildersinclude zeolites, diphosphates (pyro-phosphates), triphosphates such assodium triphosphate (STP or STPP), carbonates such as sodium car-bonate,soluble silicates such as sodium metasilicate, layered silicates (e.g.,SKS-6 from Hoechst), ethan-olamines such as 2-aminoethan-1-ol (MEA),diethanolamine (DEA, also known as iminodiethanol), trieth-anolamine(TEA, also known as 2,2′,2″-nitrilotriethanol), and carboxymethyl inulin(CMI), and combinations thereof.

The detergent composition may also comprise 0-20% by weight, such asabout 5% to about 10%, of a detergent co-builder, or a mixture thereof.The detergent composition may include a co-builder alone, or incombination with a builder, for example a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocar-boxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetri-aminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphos-phonic acid(HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA),diethylenetriamine-pentakis(methylenephosphonic acid) (DTPMPA or DTMPA),N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-mono-propionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL),N-(2-sulfoethyl)-glutamic acid (SEGO, N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(2-hydroxyethyl)-ethylidenediamine-N,N;N′-triacetate (HEDTA),diethanolgly-cine (DEG), diethylenetriamine penta(methylenephosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO 09/102854, U.S. Pat. No.5,977,053

Bleaching Systems

The detergent may comprise 0-50% by weight, such as about 0.1% to about25%, of a bleaching system. Any bleaching system known in the art foruse in laundry detergents may be utilized. Suitable bleaching systemcomponents include bleaching catalysts, photobleaches, bleachactivators, sources of hydrogen peroxide such as sodium percarbonate andsodium perborates, preformed peracids and mixtures thereof. Suitablepreformed peracids include, but are not limited to, peroxycarboxylicacids and salts, percarbonic acids and salts, perimidic acids and salts,peroxymonosulfuric acids and salts, for example, Oxone (R), and mixturesthereof. Non-limiting examples of bleaching systems includeperoxide-based bleaching systems, which may comprise, for example, aninorganic salt, including alkali metal salts such as sodium salts ofperborate (usually mono- or tetra-hydrate), percarbonate, persulfate,perphosphate, persilicate salts, in combination with a peracid-formingbleach activator. The term bleach activator is meant herein as acompound which reacts with peroxygen bleach like hydrogen peroxide toform a peracid. The peracid thus formed constitutes the activatedbleach. Suitable bleach activators to be used herein include thosebelonging to the class of esters amides, imides or anhydrides. Suitableexamples are tetracetylethylene diamine (TAED), sodium4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxydodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed inWO98/17767. A particular family of bleach activators of interest wasdisclosed in EP624154 and in that family is acetyl triethyl citrate(ATC). ATC or a short chain triglyceride like triacetin has theadvantage that it is environmental friendly as it eventually degradesinto citric acid and alcohol. Furthermore, acetyl triethyl citrate andtriacetin has a good hydrolytical stability in the product upon storageand it is an efficient bleach activator. Finally, ATC provides a goodbuilding capacity to the laundry additive. Alternatively, the bleachingsystem may comprise peroxy-acids of, for example, the amide, imide, orsulfone type. The bleaching system may also comprise peracids such as6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system may alsoinclude a bleach catalyst. In some embodiments the bleach component maybe an organic catalyst selected from the group consisting of organiccatalysts having the following formulae:

(iii) and mixtures thereof; wherein each R¹ is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, or each R¹ is independently a branchedalkyl group containing from 9 to 18 carbons or linear alkyl groupcontaining from 11 to 18 carbons, or each R¹ is independently selectedfrom the group consisting of 2-propylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hex-yldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-penta-decyl.Other exemplary bleaching systems are described, e.g. in WO2007/087258,WO2007/087244, WO2007/087259 and WO2007/087242. Suitable photobleachesmay for example be sulfonated zinc phthal-ocyanine.

Polymers

The detergent may comprise 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1% of a polymer. Any polymer known in the art for use indetergents may be utilized. The polymer may function as a co-builder asmentioned above, or may provide anti-redeposition, fiber protection,soil release, dye transfer inhibition, grease cleaning and/oranti-foaming properties. Some polymers may have more than one of theabove-mentioned properties and/or more than one of the below-mentionedmotifs. Exemplary polymers include (carboxymethyl)cellulose (CMC),poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP),poly(eth-yleneglycol) or poly(ethylene oxide) (PEG), ethoxylatedpoly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylatessuch as PAA, PAA/PMA, poly-aspartic acid, and laurylmethacrylate/acrylic acid co-polymers, hydrophobically modified CMC(HM-CMC) and silicones, copolymers of terephthalic acid and oligomericglycols, copolymers of poly(ethylene terephthalate) and poly(oxyetheneterephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI),poly(vinylpyridine-N-oxide) (PVPO or PVPNO) andpolyvinylpyrrol-idone-vinylimidazole (PVPVI). Further exemplary polymersinclude sulfonated polycarboxylates, polyeth-ylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions may also comprise fabric hueing agents suchas dyes or pigments, which when formulated in detergent compositions candeposit onto a fabric when said fabric is contacted with a wash liquorcomprising said detergent compositions and thus altering the tint ofsaid fabric through absorp-tion/reflection of visible light. Fluorescentwhitening agents emit at least some visible light. In contrast, fabrichueing agents alter the tint of a surface as they absorb at least aportion of the visible light spectrum. Suitable fabric hueing agentsinclude dyes and dye-clay conjugates, and may also include pigments.Suitable dyes include small molecule dyes and polymeric dyes. Suitablesmall molecule dyes include small molecule dyes selected from the groupconsisting of dyes falling into the Colour Index (C.I.) classificationsof Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, AcidViolet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, forexample as described in WO2005/03274, WO2005/03275, WO2005/03276 andEP1876226 (hereby incorporated by reference). The detergent compositioncomprises from about 0.00003 wt % to about 0.2 wt %, from about 0.00008wt % to about 0.05 wt %, or even from about 0.0001 wt % to about 0.04 wt% fabric hueing agent. The composition may comprise from 0.0001 wt % to0.2 wt % fabric hueing agent when the composition is in the form of aunit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO2007/087257 and WO2007/087243.

Additional Enzymes

The detergent additive as well as the detergent composition may compriseone or more [additional] enzymes such as a xanthan lyase, protease,lipase, cutinase, an amylase, lichenase, carbohydrase, cellulase,pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., alaccase, and/or peroxidase.

In general, the properties of the selected enzyme(s) should becompatible with the selected detergent, (La, pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theen-zyme(s) should be present in effective amounts.

Cellulases

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, U.S. Pat.Nos. 5,691,178, 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Example of cellulases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4) are those having described in WO02/099091.

Other examples of cellulases include the family 45 cellulases describedin WO96/29397, and especially variants thereof having substitution,insertion and/or deletion at one or more of the positions correspondingto the following positions in SEQ ID NO: 8 of WO 02/099091: 2, 4, 7, 8,10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43,44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82,84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113,114, 117, 119, 121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146,147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c,160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178,181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 20, suchas P19A, G20K, Q44K, N48E, Q119H or Q146 R.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases

The additional enzyme may be another protease or protease variant. Theprotease may be of animal, vegetable or microbial origin, includingchemically or genetically modified mutants. Microbial origin may beused. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the S1 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteasesprotease may for example be a thermolysin from e.g. family M4, M5, M7 orM8.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family. In one aspect the protease may bea subtilase, such as a subtilisin or a variant hereof. Further thesubtilases (and the serine proteases) are characterised by having twoactive site amino acid residues apart from the serine, namely ahistidine and an aspartic acid residue.

Examples of subtilisins are those derived from Bacillus such assubtilisin lentus, Bacillus lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO 89/06279 and proteasePD138 (WO 93/18140). Additional serine protease examples are describedin WO 98/020115, WO 01/44452, WO 01/58275, WO 01/58276, WO 03/006602 andWO 04/099401. An example of a subtilase variants may be those havingmutations in any of the positions: 3, 4, 9, 15, 27, 36, 68, 76, 87, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129,130, 160, 167, 170, 194, 195, 199, 205, 217, 218, 222, 232, 235, 236,245, 248, 252 and 274 using the BPN′ numbering. The subtilase variantsmay comprise the mutations: S3T, V41, S9R, A15T, K27R, *36D, V68A, N76D,N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V1041,Y,N, S106A,G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S,A194P, G195E, V199M, V2051, L217D, N218D, M222S, A232V, K235L, Q236H,Q245R, N252K, T274A (using BPN′ numbering). A further protease is thealkaline protease from Bacillus lentus DSM 5483, as described forexample in WO 95/23221, and variants thereof which are described in WO92/21760, WO 95/23221, EP 1921147 and EP 1921148.

Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO 89/06270 and WO94/25583. Examples of useful proteases are the variants described in WO92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially thevariants with substitutions in one or more of the following positions:27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218,222, 224, 235, and 274.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO 07/044993.

Non-limiting commercially available protease enzymes include Alcalase™,Coronase™, Duralase™ Durazym™, Esperase™, Everlase™, Kannase™,Liquanase™, Liquanase Ultra™, Ovozyme™, Polarzyme™, Primase™, Relase™,Savinase™ and Savinase Ultra™, (Novozymes A/S), Axapem™ (Gist-BrocasesN.V.), BLAP and BLAP X (Henkel AG & Co. KGaA), Excellase™, FN2™, FN3™,FN4™, Maxaca™, Maxapem™, Maxatase™, Properase™, Purafast™, PurafectlM,Purafect OxP™, Purafect Prime™ and Puramax™ (Genencor int.).

Lipases and Cutinases

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanu-ginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowre-named to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412), Geobacillus stearothermophiluslipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), andlipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis(WO12/137147).

Further examples are lipases sometimes referred to as acyltransferasesor perhydrolases, e.g. acyltransferases with homology to Candidaantarctica lipase A (WO10/111143), acyltransferase from Mycobacteriumsmegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279),and variants of the M. smegmatis perhydrolase in particular the S54Vvariant used in the commercial product Gentle Power Bleach from HuntsmanTextile Effects Pte Ltd (WO10/100028).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Non-limiting commercial lipase products include Lipolase™, Lipex™;Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally fromGenencor) and Lipomax (originally from Gist-Brocades).

Amylases

The amylase may be an alpha-amylase, a beta-amylase or a glucoamylaseand may be of bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Amylases include, for example,alpha-amylases obtained from Bacillus, e.g., a special strain ofBacillus licheniformis, described in more detail in GB 1,296,839.

Examples of amylases are those having SEQ ID NO: 3 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof.Non-limiting variants are described in WO 94/02597, WO 94/18314, WO97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202,207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444 of SEQ ID NO:3 in WO 95/10603.

Other amylases are variants of SEQ ID NO: 1 of WO 2016/203064 having atleast 75% sequence identity to SEQ ID NO: 1 thereof. Non-limitingvariants are variants comprising a modification in one or more positionscorresponding to positions 1, 54, 56, 72, 109, 113, 116, 134, 140, 159,167, 169, 172, 173, 174, 181, 182, 183, 184, 189, 194, 195, 206, 255,260, 262, 265, 284, 289, 304, 305, 347, 391, 395, 439, 469, 444, 473,476, or 477 of SEQ ID NO: 1, wherein said alpha-amylase variant has asequence identity of at least 75% but less than 100% to SEQ ID NO: 1.

Further amylases which can be used are amylases having SEQ ID NO: 6 inWO 02/010355 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Non-limiting variants of SEQ ID NO: 6 are those having a deletionin positions 181 and 182 and a substitution in position 193.

Other amylase examples are hybrid alpha-amylase comprising residues 1-33of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ IDNO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformisalpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having90% sequence identity thereof. Non-limiting variants of this hybridalpha-amylase are those having a substitution, a deletion or aninsertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Variants of the hybridalpha-amylase may include residues 1-33 of the alpha-amylase derivedfrom B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 andresidues 36-483 of SEQ ID NO: 4 are those having the substitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48+T49+G107+H156+A181+N190+1201+A209+Q264.

Further amylase examples are amylases having SEQ ID NO: 6 in WO99/019467 or variants thereof having 90% sequence identity to SEQ ID NO:6. Non-limiting variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, 1206, E212, E216 and K269.Non-limiting amylases are those having deletion in positions G182 andH183 or positions H183 and G184.

Additional amylases are those having SEQ ID NO: 1, SEQ ID NO: 2 or SEQID NO: 7 of WO 96/023873 or variants thereof having 90% sequenceidentity to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7. Non-limitingvariants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those havinga substitution, a deletion or an insertion in one or more of thefollowing positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260,269, 304 and 476. Non-limiting variants are those having a deletion inpositions 182 and 183 or positions 183 and 184. Non-limiting amylasevariants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those havinga deletion in positions 183 and 184 and a substitution in positions 140,195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Non-limiting variants of SEQID NO: 10 in WO 01/66712 are those having a substitution, a deletion oran insertion in one of more of the following positions: 176, 177, 178,179, 190, 201, 207, 211 and 264.

Further amylases which can be used are amylases having SEQ ID NO: 2 ofWO 09/061380 or variants thereof having 90% sequence identity to SEQ IDNO: 2. Non-limiting variants of SEQ ID NO: 2 are those having asubstitution, a deletion or an insertion in one of more of the followingpositions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182,G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359,K444 and G475. Non-limiting variants of SEQ ID NO: 2 are those havingthe substitution in one of more of the following positions: Q87E,R,Q98R, S125A, N128C, T131I , T1651, K178L, T182G, M201L, F202Y, N225E,R,N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/ordeletion in position R180 and/or S181. Non-limiting amylase variants ofSEQ ID NO: 2 are those having the substitutions:N128C+K178L+T182G+Y305R+G475K;N1280+K178L+T182G+F202Y+Y305R+D319T+G475K;S125A+N1280+K178L+T182G+Y305R+G475K; orS125A+N128C+T131I+T1651+K178L+T182G+Y305R+G475K wherein the variantoptionally further comprises a substitution at position 243 and/or adeletion at position 180 and/or position 181.

Other examples of amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90%, such as at least 95%,sequence identity to SEQ ID NO: 12. Non-limiting amylase variants arethose having a substitution, a deletion or an insertion in one of moreof the following positions of SEQ ID NO: 12 in WO01/66712: R28, R118,N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302,S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445,K446, Q449, R458, N471, N484. Non-limiting amylases include variantshaving a deletion of D183 and G184 and having the substitutions R118K,N195F, R320K and R458K, and a variant additionally having substitutionsin one or more position selected from the group: M9, G149, G182, G186,M202, T257, Y295, N299, M323, E345 and A339, non-limiting a variant thatadditionally has substitutions in all these positions.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme PIus™, Natalase™ and BAN™ (Novozymes A/S),Rapidase™ and Purastar™ (from Genencor International Inc.).

Peroxidases/Oxidases

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,i.e., a separate additive or a combined additive, can be formulated, forexample, as a granulate, liquid, slurry, etc. Non-limiting detergentadditive formulations are granulates, in particular non-dustinggranulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants:

The detergent compositions can also contain dispersants. In particularpowdered detergents may comprise dispersants. Suitable water-solubleorganic materials include the homo- or co-polymeric acids or theirsalts, in which the polycarboxylic acid comprises at least two carboxylradicals separated from each other by not more than two carbon atoms.Suitable dispersants are for example described in Powdered Detergents,Surfactant science series volume 71, Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents:

The detergent compositions may also include one or more dye transferinhibiting agents. Suitable polymeric dye transfer inhibiting agentsinclude, but are not limited to, polyvinylpyrrolidone polymers,polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone andN-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a subject composition, the dyetransfer inhibiting agents may be present at levels from about 0.0001%to about 10%, from about 0.01% to about 5% or even from about 0.1% toabout 3% by weight of the composition.

Fluorescent Whitening Agent:

The detergent compositions will also contain additional components thatmay tint articles being cleaned, such as fluorescent whitening agent oroptical brighteners. Where present the brightener is at a level of about0,01% to about 0,5%. Any fluorescent whitening agent suitable for use ina laundry detergent composition may be used in the composition. The mostcommonly used fluorescent whitening agents are those belonging to theclasses of diaminostilbene-sulphonic acid derivatives, diarylpy-razolinederivatives and bisphenyl-distyryl derivatives. Examples of thediaminostilbene-sulphonic acid derivative type of fluorescent whiteningagents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-ani-lino-s-triazin-6-ylamino)-stilbene-2,2′-disulphonate;4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)-stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)-stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4-(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)-stilbene-2,2′-disulphonateand 2-(stilbyl-4″-naptho-1,2′:4,5)-1,2,3-trizole-2″-sulphonate.Non-limiting fluorescent whitening agents are Tinopal DMS and TinopalCBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is thedisodium salt of 4,4′-bis-(2-morpho-lino-4anilino-s-triazin-6-ylamino)-stilbene disulphonate. Tinopal CBS is thedisodium salt of 2,2′-bis-(phenyl-styryl) disulphonate. Also usable arefluorescent whitening agents is the commercially available Parawhite KX,supplied by Paramount Minerals and Chemicals, Mumbai, India. Otherfluorescers suitable for use include the 1-3-diaryl pyrazolines and the7-alkylaminocoumarins. Suitable fluorescent brightener levels includelower levels of from about 0.01, from 0.05, from about 0.1 or even fromabout 0.2 wt % to upper levels of 0.5 or even 0.75 wt %.

Soil Release Polymers:

The detergent compositions may also include one or more soil releasepolymers which aid the removal of soils from fabrics such as cotton andpolyester based fabrics, in particular the removal of hydrophobic soilsfrom polyester based fabrics. The soil release polymers may for examplebe non-ionic or anionic terephthalate-based polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyesterpolyamides see for example Chapter 7 in Powdered Detergents, Surfactantscience series volume 71, Marcel Dekker, Inc. Another type of soilrelease polymers are amphiphilic alkoxylated grease cleaning polymerscomprising a core structure and a plurality of alkoxylate groupsattached to that core structure. The core structure may comprise apolyalkylenimine structure or a polyalkanolamine structure as describedin detail in WO 2009/087523 (hereby incorporated by reference).Furthermore, random graft co-polymers are suitable soil release polymersSuitable graft co-polymers are described in more detail in WO2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated byreference). Other soil release polymers are substituted polysaccharidestructures especially substituted cellulosic structures such as modifiedcellulose derivatives such as those described in EP 1867808 or WO2003/040279 (both are hereby incorporated by reference). Suitablecellulosic polymers include cellulose, cellulose ethers, celluloseesters, cellulose amides and mixtures thereof. Suitable cellulosicpolymers include anionically modified cellulose, nonionically modifiedcellulose, cationically modified cellulose, zwitterionically modifiedcellulose, and mixtures thereof. Suitable cellulosic polymers includemethyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxylethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methylcellulose, and mixtures thereof.

Anti-Redeposition Agents:

The detergent compositions may also include one or moreanti-redeposition agents such as carboxymethylcellulose (CMC), polyvinylalcohol (PVA), polyvinylpyrrolidone (PVP), poly-oxyethylene and/orpolyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers ofacrylic acid and maleic acid, and ethoxylated polyethyleneimines. Thecellulose-based polymers described under soil release polymers above mayalso function as anti-redeposition agents.

Other Suitable Adjunct Materials Include, but are not Limited to,Anti-Shrink Agents, Anti-Wrinkling Agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

Formulation of Detergent Products

The detergent composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a pouch havingone or more compartments, a regular or compact powder, a granule, apaste, a gel, or a regular, compact or concentrated liquid. There are anumber of detergent formulation forms such as layers (same or differentphases), pouches, as well as forms for machine dosing unit.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition from the pouchprior to water contact. The pouch is made from water soluble film whichencloses an inner volume. Said inner volume can be divided intocompartments of the pouch. Non-limiting films are polymeric materials,such as polymers which are formed into a film or sheet. Non-limitingpolymers, copolymers or derivates therof are selected polyacrylates, andwater-soluble acrylate copolymers, methyl cellulose, carboxy methylcellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, suchas polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose(HPMC). In a non-limiting embodiment, the level of polymer in the filmfor example PVA is at least about 60%. Non-limiting average molecularweight will typically be about 20,000 to about 150,000. Films can alsobe of blend compositions comprising hydrolytically degradable andwater-soluble polymer blends such as polyactide and polyvinyl alcohol(known under the Trade reference M8630 as sold by Chris Craft In. Prod.Of Gary, Ind., US) plus plasticisers like glycerol, ethylene glycerol,Propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by thewater-soluble film. The compartment for liquid components can bedifferent in composition than compartments containing solids. Ref:(US2009/0011970 A1).

Detergent ingredients can be separated physically from each other bycompartments in water dissolv-able pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent. A liquid or geldetergent may be non-aqueous.

Laundry Soap Bars

The enzymes may be added to laundry soap bars and used for hand washinglaundry, fabrics and/or textiles. The term laundry soap bar includeslaundry bars, soap bars, combo bars, syndet bars and detergent bars. Thetypes of bar usually differ in the type of surfactant they contain, andthe term laundry soap bar includes those containing soaps from fattyacids and/or synthetic soaps. The laundry soap bar has a physical formwhich is solid and not a liquid, gel or a powder at room temperature.The term solid is defined as a physical form which does notsignificantly change over time, i.e. if a solid object (e.g. laundrysoap bar) is placed inside a container, the solid object does not changeto fill the container it is placed in. The bar is a solid typically inbar form but can be in other solid shapes such as round or oval.

The laundry soap bar may contain one or more additional enzymes,protease inhibitors such as peptide aldehydes (or hydrosulfite adduct orhemiacetal adduct), boric acid, borate, borax and/or phenylboronic acidderivatives such as 4-formylphenylboronic acid, one or more soaps orsynthetic surfactants, polyols such as glycerine, pH controllingcompounds such as fatty acids, citric acid, acetic acid and/or formicacid, and/or a salt of a monovalent cation and an organic anion whereinthe monovalent cation may be for example Na⁺, K⁺ or NH₄ ⁺ and theorganic anion may be for example formate, acetate, citrate or lactatesuch that the salt of a monovalent cation and an organic anion may be,for example, sodium formate.

The laundry soap bar may also contain complexing agents like EDTA andHEDP, perfumes and/or different type of fillers, surfactants e.g.anionic synthetic surfactants, builders, polymeric soil release agents,detergent chelators, stabilizing agents, fillers, dyes, colorants, dyetransfer inhibitors, alkoxylated polycar-bonates, suds suppressers,structurants, binders, leaching agents, bleaching activators, clay soilremoval agents, anti-redeposition agents, polymeric dispersing agents,brighteners, fabric softeners, perfumes and/or other compounds known inthe art.

The laundry soap bar may be processed in conventional laundry soap barmaking equipment such as but not limited to: mixers, plodders, e.g. atwo-stage vacuum plodder, extruders, cutters, logo-stampers, coolingtunnels and wrappers. The invention is not limited to preparing thelaundry soap bars by any single method. The premix may be added to thesoap at different stages of the process. For example, the premixcontaining a soap, an enzyme, optionally one or more additional enzymes,a protease inhibitor, and a salt of a monovalent cation and an organicanion may be prepared, and the mixture is then plodded. The enzyme andoptional additional enzymes may be added at the same time as theprotease inhibitor for example in liquid form. Besides the mixing stepand the plodding step, the process may further comprise the steps ofmilling, extruding, cutting, stamping, cooling and/or wrapping.

Method of Producing the Composition

The method may be relevant for the (storage) stability of the detergentcomposition: e.g. Soap bar premix method WO2009155557.

Uses

The detergent compositions may be used for example in any detergentapplication which requires the degradation of xanthan gum.

Use to Degrade Xanthan Gum

Xanthan gum has been used as an ingredient in many consumer productsincluding foods and cosmetics and has found use in the oil industry.Therefore, the degradation of xanthan gum can result in improvedcleaning processes, such as the easier removal of stains containinggums, such as xanthan gum. Thus, use of detergent compositionscomprising GH9 endoglucanases (e.g. variants described herein) maydegrade xanthan gum. The use of xanthan lyases in the detergentcompositions may degrade xanthan gum. An embodiment is the use of GH9endoglucanases as described herein (e.g. variants) together with xanthanlyases in detergent compositions to degrade xanthan gum. Degradation ofxanthan gum can be measured using the viscosity reduction assay (e.g.,ViPr assay) or alternatively as described in example 3 herein.

GH9 endoglucanase activity may alternatively be measured by assessmentof reducing ends on xanthan gum pre-treated with xanthan lyase using thecolorimetric assay developed by Lever (1972), Anal. Biochem. 47:273-279, 1972. A non-limiting embodiment is the use of 0.1% xanthan gumpre-treated with xanthan lyase. Degradation of xanthan gum pre-treatedwith xanthan lyase may be determined by calculating difference betweenblank and sample, wherein a difference of more than 0.5 mAU, more than0.6 mAU, more than 0.7 mAU or more than 0.8 mAU, shows degradation ofxanthan gum pre-treated with xanthan lyase.

Xanthan lyase activity may alternatively be measured by assessment ofreducing ends liberated from xanthan gum using the colorimetric assaydeveloped by Lever (1972), Anal. Biochem. 47: 273-279, 1972. Anon-limiting embodiment is the use of 0.1% xanthan gum. Degradation ofxanthan gum may be determined by calculating difference between blankand sample wherein a difference of more than 0.1 mAU, more than 0.15mAU, more than 0.2 mAU or more than 0.25 mAU shows degradation ofxanthan gum.

GH9 endoglucanase and xanthan lyase activity may alternatively bemeasured by assessment of reducing ends liberated from xanthan gum usingthe colorimetric assay developed by Lever (1972), Anal. Biochem. 47:273-279, 1972. A non-limiting embodiment is the use of 0.1% xanthan gum.Degradation of xanthan gum may be determined by calculating differencebetween blank and sample wherein a difference of more than 0.4 mAU, morethan 0.5 mAU, more than 0.6 mAU or more than 0.8 mAU shows degradationof xanthan gum.

The invention also relates to methods for degrading xanthan gumcomprising applying a detergent composition comprising one or more GH9endoglucanases as described herein (e.g. variants) to xanthan gum. Anembodiment is a method for degrading xanthan gum comprising applying adetergent composition comprising one or more GH9 endoglucanases asdescribed herein (e.g. variants) together with one or more xanthanlyases to xanthan gum.

Use in Detergents

GH9 endoglucanases as described herein may be used in detergentcompositions for cleaning processes such as the laundering of textilesand fabrics (e.g. household laundry washing and industrial laundrywashing), as well as household and industrial hard surface cleaning,such as dish wash. The GH9 endoglucanases as described herein may beadded to a detergent composition comprising of one or more detergentcomponents.

In some aspects, GH9 endoglucanases as described herein may be usedtogether with a xanthan lyase(s) in detergent compositions for cleaningprocesses such as the laundering of textiles and fabrics (e.g. householdlaundry washing and industrial laundry washing), as well as householdand industrial hard surface cleaning, such as dish wash. The GH9endoglucanases as described herein together with a xanthan lyase(s) maybe added to a detergent composition comprising of one or more detergentcomponents.

The polypeptides described herein (e.g. variants) may be added to andthus become a component of a detergent composition. The detergentcomposition may be formulated, for example, as a hand or machine laundrydetergent composition for both household and industrial laundrycleaning, including a laundry additive composition suitable forpre-treatment of stained fabrics and a rinse added fabric softenercomposition, or be formulated as a detergent composition for use ingeneral household or industrial hard surface cleaning operations, or beformulated for hand or machine (both household and industrial)dishwashing operations. In a specific aspect, a detergent additive mayinclude a polypeptide as described herein.

The invention also relates to methods for degrading xanthan gum on thesurface of a textile or hard surface, such as dish wash, comprisingapplying a detergent composition comprising one or more GH9endoglucanases as described herein (e.g. variants) to xanthan gum. Insome aspects, the invention relates a method for degrading xanthan gumon the surface of a textile or hard surface, such as dish wash,comprising applying a detergent composition comprising one or more GH9endoglucanases as described herein (e.g. variants) together with one ormore xanthan lyases to xanthan gum. In some aspects, the inventionrelates to a detergent composition comprising one or more detergentcomponents as described herein. Also encompassed is the use of GH9endoglucanases having an enzyme detergency benefit in the detergentcompositions.

It has been contemplated that the use of a GH9 endoglucanase asdescribed herein (e.g., a) alone gives an enzyme detergency benefit, anenzyme detergency benefit on xanthan gum.

In some aspects, the invention relates to the use of a detergentcomposition comprising one or more detergent components and an isolatedGH9 endoglucanase as described herein (e.g. a variant) together with axanthan lyase. In some aspects, the invention relates to the use of adetergent composition comprising one or more detergent components and anisolated GH9 endoglucanase (e.g. a variant) together with a xanthanlyase.

-   1. A detergent composition comprising an endoglucanase variant,    comprising an alteration (e.g., a substitution, deletion or    insertion) at one or more positions in a region selected from the    group consisting of:-   i) region 1 corresponding to amino acids 95 to 105 of SEQ ID NO: 2,    e.g., said alteration at one or more positions selected from the    group consisting of positions: 95, 96, 97, 98, 99, 100, 101, 102,    103, 104, 105, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2),-   ii) region 2 corresponding to amino acids 115 to 138 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 115, 116, 117, 118, 119, 120, 121,    122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,    135, 136, 137, 138, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2),-   iii) region 3 corresponding to amino acids 210 to 251 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 210, 211, 212, 213, 214, 215, 216,    217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,    230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,    243, 244, 245, 246, 247, 248, 249, 250, 251, wherein said positions    correspond to amino acid positions of SEQ ID NO: 2 (e.g., using the    numbering of SEQ ID NO: 2),-   iv) region 4 corresponding to amino acids 267 to 301 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 267, 268, 269, 270, 271, 272, 273,    274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,    300, 301, wherein said positions correspond to amino acid positions    of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO: 2),-   v) region 5 corresponding to amino acids 339 to 361 of SEQ ID NO: 2,    e.g., said alteration at one or more positions selected from the    group consisting of positions: 339, 340, 341, 342, 343, 344, 345,    346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,    359, 360, 361, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2),-   vi) region 6 corresponding to amino acids 547 to 595 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 547, 548, 549, 550, 551, 552, 553,    554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,    567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579,    580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592,    593, 594, 595, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2),-   vii) region 7 corresponding to amino acids 612 to 660 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 612, 613, 614, 615, 616, 617, 618,    619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631,    632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644,    645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657,    658, 659, 660, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2),-   viii) region 8 corresponding to amino acids 806 to 828 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 806, 807, 808, 809, 810, 811, 812,    813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825,    826, 827, 828, wherein said positions correspond to amino acid    positions of SEQ ID NO: 2 (e.g., using the numbering of SEQ ID NO:    2), and-   ix) region 9 corresponding to amino acids 839 to 1042 of SEQ ID NO:    2, e.g., said alteration at one or more positions selected from the    group consisting of positions: 839, 840, 841, 842, 843, 844, 845,    846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858,    859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871,    872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884,    885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897,    898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910,    911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923,    924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936,    937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949,    950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962,    963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975,    976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988,    989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001,    1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012,    1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023,    1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034,    1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, wherein said    positions correspond to amino acid positions of SEQ ID NO: 2 (e.g.,    using the numbering of SEQ ID NO: 2),    -   wherein said variant has at least 60%, e.g., at least 65%, at        least 70%, at least 75%, at least 80%, at least 85%, at least        90%, at least 95%, at least 96%, at least 97%, at least 98%, or        at least 99%, and less than 100% sequence identity to SEQ ID NO:        2; preferably said endoglucanase variant has activity on xanthan        gum pre-treated with xanthan lyase, further preferably said        activity is a xanthan gum degrading activity.-   2. The detergent composition comprising an endoglucanase variant of    paragraph 1, which is a variant of a parent endoglucanase selected    from the group consisting of:    -   a) a polypeptide having at least 60% sequence identity to the        mature polypeptide of SEQ ID NO: 2;    -   b) a polypeptide encoded by a polynucleotide that hybridizes        under low stringency conditions with (i) the mature polypeptide        coding sequence of SEQ ID NO: 1, or (ii) the full-length        complement of (i);    -   c) a polypeptide encoded by a polynucleotide having at least 60%        identity to the mature polypeptide coding sequence of SEQ ID NO:        1; and    -   d) a fragment of the mature polypeptide of SEQ ID NO: 2, which        has endoglucanase activity.-   3. The detergent composition comprising an endoglucanase variant of    paragraph 2, wherein the parent endoglucanase having at least 60%,    e.g., at least 65%, at least 70%, at least 75%, at least 80%, at    least 85%, at least 90%, at least 95%, at least 96%, at least 97%,    at least 98%, at least 99% or 100% sequence identity to the mature    polypeptide of SEQ ID NO: 2.-   4. The detergent composition comprising an endoglucanase variant of    any of paragraphs 2-3, wherein the parent endoglucanase is encoded    by a polynucleotide that hybridizes under low stringency conditions,    medium stringency conditions, medium-high stringency conditions,    high stringency conditions, or very high stringency conditions    with (i) the mature polypeptide coding sequence of SEQ ID NO: 1    or (ii) the full-length complement of (i).-   5. The detergent composition comprising an endoglucanase variant of    any of paragraphs 2-4, wherein the parent endoglucanase is encoded    by a polynucleotide having at least 60%, e.g., at least 65%, at    least 70%, at least 75%, at least 80%, at least 85%, at least 90%,    at least 95%, at least 96%, at least 97%, at least 98%, at least    99%, or 100% sequence identity to the mature polypeptide coding    sequence of SEQ ID NO: 1.-   6. The detergent composition comprising an endoglucanase variant of    any of paragraphs 2-5, wherein the parent endoglucanase comprises or    consists of the mature polypeptide of SEQ ID NO: 2.-   7. The detergent composition comprising an endoglucanase variant of    any of paragraphs 2-6, wherein the parent endoglucanase is a    fragment of the mature polypeptide of SEQ ID NO: 2, wherein the    fragment has endoglucanase activity.-   8. The detergent composition comprising an endoglucanase variant of    any of paragraphs 2-7, which has at least 60%, e.g., at least 65%,    at least 70%, at least 75%, at least 80%, at least 85%, at least    90%, at least 95% identity, at least 96%, at least 97%, at least    98%, or at least 99%, but less than 100%, sequence identity to the    amino acid sequence of the parent endoglucanase.-   9. The detergent composition comprising an endoglucanase variant of    any of paragraphs 1-8, wherein said variant has at least 61%, at    least 62%, at least 63%, at least 64%, at least 65%, at least 66%,    at least 67%, at least 68%, at least 69%, at least 70%, at least    71%, at least 72%, at least 73%, at least 74%, at least 75%, at    least 76%, at least 77%, at least 78%, at least 79%, at least 80%,    at least 81%, at least 82%, at least 83%, at least 84%, at least    85%, at least 86%, at least 87%, at least 88%, at least 89%, at    least 90%, at least 91%, at least 92%, at least 93%, at least 94%,    at least 95%, at least 96%, at least 97%, at least 98%, or at least    99% sequence identity to SEQ ID NO: 2.-   10. The detergent composition comprising an endoglucanase variant of    any of paragraphs 1-9, wherein said alteration at one or more    positions is selected from the group consisting of alterations in    positions: 17, 20, 51, 53, 55, 56, 60, 63, 79, 87, 186, 192, 302,    311, 313, 387, 388, 390, 403, 408, 410, 416, 448, 451, 471, 472,    476, 489, 507, 512, 515, 538, 598, 599, 602, 603, 605, 609, 676,    688, 690, 694, 697, 698, 699, 711, 719, 754, 756, 760, 781, 786,    797, 833, 834, 835 and 1048 of SEQ ID NO: 2, wherein numbering is    according to SEQ ID NO: 2.-   11. The detergent composition comprising an endoglucanase variant of    any of paragraphs 1-10, wherein said alteration at one or more    positions is selected from the group consisting of: 517A, F20P,    F20N, F20G, F20Y, K51Q, K51H, E53P, E53G, Y55M, V56M, Y60F, S63F,    T87R, A186P, K192N, 1302D, I302H, 1302V, 1302M, H311N, S313D, I387T,    K388R, K390Q, 1403Y, E408D, E408S , E408P, E408A, E408G, E408N,    P410G, Q416S, Q416D, A448E, A448W, A448S, K451S, G471S, S472Y,    D476R, Q489P, K507R, K512P, S515V, S538C, Y579W, S598Q, A599S,    1602T, 1602D, V603P, S605T, G609E, D676H, A688G, Y690F, T694A,    T697G, R698W, T699A, T711V, T711Y, W719R, K754R, V756H, V756Y,    S760G, T781M, N786K, T797S, A824D, N833D, Q834E, S835D and F1048W of    SEQ ID NO: 2.-   12. The detergent composition comprising an endoglucanase variant    according to any one of paragraphs 1-11, wherein the variant further    comprises an alteration at one or more positions selected from the    group consisting of alterations in positions: 216, 283, 346, 559,    564, 579, 636, 638, 651, 824, 848, 869, 880, 881, 883, 887, 892,    905, 906, 912, 921, 928, 934, 937, 948, 956, 999 and 1037.-   13. The detergent composition comprising an endoglucanase variant    according to any one of paragraphs 1-12, further wherein said    alteration at one or more positions is selected from the group    consisting of: N216D, N216Q, A283D, A346D, A559P, A559N, A564E,    Y579W, S636K, S636N, F638N, A651P, A824D, N848D, A869V, R880K,    V881T, V881Q, T883R, T887K, T892P, N905D, F906A, A912V, K921R,    S928D, Y934G, A937E, K948R, Q956Y, T999R, and A1037E.-   14. The detergent composition comprising an endoglucanase variant    according to any one of claims 1-13, wherein the variant comprises    one or more of the following set of substitutions:

F20Y + A448S + T781M E408D + K451S E408D + A448E E408D Y579W + E408D

-   15. The detergent composition comprising an endoglucanase variant    according to any one of claims 1-14, wherein the variant comprises    one or more of the following set of substitutions:

S17A, F20P, N216D, A283D, H311N, E408D, Y579W, I602T, A651P, A688G,T883R, F906A, Y934G, Q956Y F20P, I302D, S313D, E408D, D476R, Y579W,S636K, T697G, V756Y, V881Q, T887K, F906A, A937E F20P, S313D, E408D,Y579W, S636K, A688G, T697G, N905D, A937E F20P, I302D, S313D, E408D,D476R, Y579W, S636K, T697G, W719R, V756Y, V881Q, T887K, F906A, A937EN216Q, S313D, E408D, D476R, Y579W, I602T, F638N, A651P, T697G, W719R,R880K, T887K, K921R, Y934G N216D, S313D, E408D, D476R, A564E, Y579W,I602T, F638N, A651P, Y690F, T697G, W719R, V756H, N833D, A869V, R880K,V881T, T887K, K921R, S928D, Y934G, T999R F20P, I302D, S313D, E408D,D476R, Y579W, S636K, T697G, W719R, V756Y, N848D, A869V, V881Q, T887K,N905D, F906A, Q912V, A937E, T999R, F1048W F20P, I302D, S313D, E408D,D476R, Q489P, Y579W, S636N, T697G, W719R, V756Y, A824D, N848D, V881Q,T887K, F906A, S928D, A937E F20P, I302D, S313D, E408D, Q416S, D476R,Q489P, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, A824D, N833D,N848D, T883R, T887K, F906A, A937E F20P, A186P, I302D, S313D, E408D,D476R, Q489P, Y579W, A599S, I602T, S636K, A651P, T697G, W719R, V756Y,N848D, T883R, T887K, F906A, A937E N216D, S313D, E408D, D476R, Y579W,I602T, V603P, F638N, A651P, A688G, T697G, W719R, V756H, R880K, T887K,K921R, S928D, Y934G, K948R F20P, K51Q, I302D, S313D, E408D, D476R,Q489P, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, N848D, T883R,T887K, F906A, A937E F20P, I302D, S313D, A346D, E408D, D476R, Q489P,Y579W, S636N, T697G, W719R, V756Y, A824D, N848D, V881Q, T887K, F906A,A937E, T999R F20P, I302D, S313D, E408D, D476R, Q489P, Y579W, I602T,S636N, T697G, W719R, V756Y, A824D, N848D, V881Q, T887K, N905D, F906A,A937E, T999R, A1037E, F1048W F20P, K51Q, I302D, S313D, E408D, D476R,Q489P, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, A824D, N848D,T883R, T887K, F906A, S928D, A937E, A1037E N216D, S313D, A346D, E408D,D476R, Q489P, A559P, Y579W, I602T, F638N, A651P, A688G, T697G, W719R,V756H, R880K, T887K, K921R, S928D, Y934G F20P, I302D, S313D, A346D,E408D, D476R, Q489P, Y579W, I602T, T697G, W719R, V756Y, N848D, V881Q,T887K, F906A, A937E N216D, S313D, E408D, D476R, Q489P, A559P, Y579W,I602T, F638N, A651P, A688G, T697G, W719R, V756H, Q834E, R880K, T887K,T892P, K921R, S928D, Y934G F20P, I302D, S313D, E408D, D476R, Q489P,A559N, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, N848D, T883R,T887K, F906A, S928D, A937E F20P, I302D, S313D, E408D, D476R, Q489P,Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, N848D, T883R, T887K,F906A, A937E F20P, I302D, S313D, E408D, Q416S, D476R, Q489P, A559N,Y579W, I602T, S636K, A651P, T697G, W719R, V756Y, N848D, T883R, T887K,F906A, A937E F20P, I302D, S313D, E408D, D476R, Y579W, I602T, S636N,T697G, W719R, V756Y, A824D, N848D, V881Q, T887K, F906A, S928D, A937E,T999R, F1048W

-   16. The detergent composition comprising an endoglucanase variant of    any of paragraphs 1-15, wherein the total number of alterations    compared to the parent endoglucanase (e.g., SEQ ID NO: 2) is between    1 and 25, e.g., between 1 and 20, e.g., between 1 and 10 or between    1 and 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.-   17. The detergent composition comprising an endoglucanase variant of    any of paragraphs 1-16, wherein said activity on xanthan gum    pre-treated with xanthan lyase is a xanthan degrading activity,    preferably said xanthan degrading activity is endoglucanase EC    3.2.1.4 activity.-   18. The detergent composition comprising an endoglucanase variant of    any of paragraphs 1-17, wherein said variant has an improved    stability in a detergent composition compared to a parent    endoglucanase (e.g., with SEQ ID NO: 2).-   19. The detergent composition comprising an endoglucanase variant of    any of paragraphs 1-18, wherein said variant has a half-life    improvement factor (HIF) of 1.0; preferably said variant has a    half-life improvement factor (HIF) of >1.0 relative to a parent    endoglucanase, e.g. an endoglucanase of SEQ ID NO: 2.-   20. The detergent composition comprising an endoglucanase variant of    paragraph 19, wherein said half-life improvement factor (HIF) is    determined after incubation of said endoglucanase variant in a    detergent composition at 25° C. for a time period from about 5 to    about 140 hours.-   21. The detergent composition of any of paragraphs 1-20, further    comprising one or more detergent components.-   22. The detergent composition of any of paragraphs 1-21 further    comprising one or more additional enzymes selected from the group    consisting of: endoglucanases, proteases, amylases, lichenases,    lipases, cutinases, cellulases, xanthan lyases, xyloglucanases,    pectinases, pectin lyases, xanthanases, peroxidases,    haloperoxygenases, catalases and mannanases, or any mixture thereof.-   23. The detergent composition of any of paragraphs 1-22, further    comprising an endoglucanase, such as xanthan endoglucanase.-   24. The detergent composition of any of paragraphs 1-23, wherein    said composition is in form of a bar, a homogenous tablet, a tablet    having two or more layers, a pouch having one or more compartments,    a regular or compact powder, a granule, a paste, a gel, or a    regular, compact or concentrated liquid.-   25. Use of a detergent composition of any of paragraphs 1-24 for    degrading xanthan gum.-   26. The use of paragraph 25, wherein said endoglucanase variant has    an enzyme detergency benefit.-   27. A method for degrading xanthan gum comprising: applying a    detergent composition of any of paragraphs 1-24 to a xanthan gum.-   28. The method of paragraph 27, wherein said xanthan gum is on a    surface or hard surface.

EXAMPLES Example 1: Construction of GH9 Endoglucanase Variants of theMature Parent Endoglucanase Having SEQ ID NO: 2

A linear integration vector-system was used for cloning of the matureparent nucleotide sequence having SEQ ID NO: 1 (same also disclosed asmature peptide within SEQ ID NO: 1 of WO 2013/167581A1) coding for themature parent polypeptide of the GH9 endoglucanase of SEQ ID NO: 2, andits variants. The linear integration construct was a PCR fusion made byfusing the gene between two Bacillus subtilis homologous chromosomalregions along with strong promoters and a chloramphenicol resistancemarker. The fusion was made by Splicing by Overlap Extension (SOE) PCR(Horton, R. M, Hunt, H. D., Ho, S. N., Pullen, J. K. and Pease, L. R.(1989). Engineering hybrid genes without the use of restriction enzymes,or gene splicing were produced by overlap extension (Gene 77: 61-68).The SOE PCR method is also described in patent application WO2003/095658. The gene was expressed under the control of a triplepromoter system (as described in WO 99/43835), consisting of thepromoters from Bacillus licheniformis alpha-amylase gene (amyL),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillusthuringiensis cryIIIA promoter including stabilizing sequence. The genecoding for chloramphenicol acetyltransferase was used as marker(described in e.g. Diderichsen, B.; Poulsen, G. B.; Joergensen, S.T.; Auseful cloning vector for Bacillus subtilis. Plasmid 30:312, 1993). Thefinal gene constructs were integrated on the Bacillus chromosome byhomologous recombination into the pectate lyase locus. The genefragments were amplified from chromosomal DNA of the correspondingstrains with gene specific primers containing overhang to the twoflanking vector fragments. All genes were expressed with a Bacilluslicheniformis alpha-amylase secretion signal having the nucleotidesequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4replacing the native secretion signal.

Variants of the mature parent GH9 endoglucanase from Paenibacillussp-62047 having SEQ ID NO: 2 as described in Examples 3-4 below weremade by the megaprimer mutagenesis method using specifically designedmutagenic oligonucleotides introducing desired mutations in theresulting sequence. Design and production methods for such mutagenicoligonucleotides introducing desired mutations into target sequences arewell known to those skilled in the art. Consequently, mutagenic oligoswere designed and synthesized corresponding to the DNA sequence flankingthe desired site(s) of mutation, separated by the DNA base pairsdefining the substitutions. The final expression cassette composed thereference GH9 endocluconase from Paenibacillus sp-62047 as describedabove (i.e., parent GH9 endocluconase having SEQ ID NO: 1). Successfulintroduction of the desired substitutions was confirmed by DNAsequencing of the GH9 endoglucanase gene. An aliquot of the PCR productwas subsequently transformed into Bacillus subtilis. Transformants wereselected on LB agar plates supplemented with 10 mM K2PO4, 0.4% extraglucose and 6 μg of chloramphenicol per ml. The resulting recombinantBacillus subtilis clone containing the integrated expression constructwas grown in liquid culture as described below. The enzyme containingsupernatants were harvested and the enzymes (variants) were eitherstress tested using a reducing sugar assay or purified as describedbelow.

Variants above were produced by fermentation using standard protocols(TB-glycerol media containing a standard trace metal mix as described inF. William Studier, “Protein production by auto-induction inhigh-density shaking cultures”, Protein Expression and Purification, 41(2005) 207-234) and grown for 4 days at 30° C. before harvested).Supernatants of samples used for stress testing were inoculated from anovernight culture grown at 37° C. and subsequently fermented in 96-wellplate format (TB-glycerol media described above without calcium in thetrace metal mix for 4 days 30° C.).

Example 2: Purification of GH9 Endoglucanase Variants

The culture broth was centrifuged at 13′000 rpm (45 min, 18° C.,F12S-6×500 rotor) using a Sorval RC-6 plus centrifuge (ThermoFisherScientific). The supernatant was supplemented with (NH₄)₂SO₄ to a finalconcentration of 0.8 M. The mixture was filtered using 0.2 μm bottle-toprapid flow filters (Nalgene). The mixture was loaded on a 50 mL PhenylSepharose High Performance (GE Healthcare, Uppsala, Swe-den)pre-equilibrated with 20 mM Tris-HCl, pH 8.0 with 0.8 M (NH₄)₂SO₄.Flowrate was set to 3 mL/min. After protein loading, the flow rate wasincreased to 5 mL/min and unbound or loosely bound protein was washedout by several column volumes of equilibration buffer. Elution wascarried out by step-wise increase of elution buffer (20 mM Tris-HCl, pH8.0). The target protein eluted during the (75-100%) elution step.Fractions of 8 mL were collected during the purification. The fractionswere evaluated using SDS-PAGE (NuPAGE, Invitrogen). Fractions elutingwith 20 mM Tris-HCl, pH 8.0 were pooled and desalted on a 350 mL G25desalting column pre-equilibrated with 20 mM Tris-HCl, pH 8.5. Thedesalted protein solution was applied on a 20 mL Source15Q columnpre-equilibrated with 20 mM Tris-HCl, pH 8.5 at 2 mL/min. Unbound orloosely bound proteins were washed using at least two column volumes ofequilibration buffer until a stable UV baseline was obtained. The flowrate was raised to 4 mL/min and elution was done by a linear NaClgradient using the elution buffer (20 mM Tris-HCl, pH 8.5+750 mM NaCl).3 mL fractions were collected during the purification. SDS-PAGE was usedto evaluate the fractions. Pure fractions were pooled and concentratedif necessary using Vivaspin 20 (10 kDa Cut-off, Sartorius). Proteinconcentration was determined using absorbance measurements at 280 nm.

Example 3: Detergent Stability Assay

GH9 endoglucanase (EG) activity (EC 3.2.1.4) was determined by reducingends on xanthan gum pre-treated with xanthan lyase using thecolorimetric assay developed by Lever (1972), Anal. Biochem. 47:273-279, 1972. Pre-treated xanthan gum is a modified form of the xanthansugar, where the terminal pyruvated mannose from side chains is removed(prepared according to Nankai et al. (1999) from the source Keltran).The GH9 mature parent endoglucanase and its variants cleave atbeta(1,4)-glucosyl bonds in the glucan backbone of pre-treated xanthangum releasing glucans with a reducing end which can be determined byreaction with p-Hydroxybenzoic acid hydrazide (PAHBAH). The increase ofcolour is proportional to the enzyme activity under the conditions usedin the assay (e.g., Table 1) and used to estimate the residual activity(RA), half-life (T112) and the half-life improvement factor (HIF).

TABLE 1 Description of assays Stress assay: Detergent Persil UniversalGel Assay buffer (AB) 50 mM MOPS, 4 mM CaCl₂, 0.01% Triton X-100, pH 7.0Reference sample conditions 4° C. for 5-138 hours Stress conditions 25,26, 28 or 30° C. for 5-138 hours Activity assay: Substrate concentration4 mg/mL modified xanthan gum Xanthan gum incubation 50° C. for 1 hPAHBAH solution 15 mg/mL 4-hydroxybenzoic acid hydrazide (PAHBAH), 50g/L potassium sodium tartrate tetrahydrate, 20 g/L NaOH PAHBAHdevelopment 95° C. for 10 min

Method Steps:

30 μL enzyme sample (purified variant, 10-150 ppm) was mixed with 270 μLdetergent using magnetic stirring for 15 minutes in a micro titer plate(MTP). This plate was designated as the “stress MTP”. 20 μL of themixture was transferred to a new MTP and diluted 100-fold using a 2-stepdilution (2×10-fold dilution). The sample was diluted into assay buffer(AB): 50 mM MOPS, 4 mM CaCl₂), 0.01% Triton X-100, pH 7.0. This dilutedMTP is the “reference MTP” and is stored at 4° C. for 5-138 h (at a timeinterval equal to that of the stress MTP below). The stress MTP wasincubated at 25, 26, 28 or 30° C. for 5-138 h. After incubation, thestress MTP was initially mixed by magnetic stirring for 15 minutes, andthe stress MTP was then diluted 100-fold as described for the referenceMTP. To assess the enzymatic activity, 50 μL of diluted enzyme:detergentsample (from both reference and stress MTPs) was mixed with 50 μL 4mg/mL modified xanthan gum in PCR plates. The samples were thenincubated at 50° C. for 1 h. Finally, the level of reducing ends wasestimated by adding 75 μL PAHBAH solution (15 mg/mL PAHBAH, 50 g/Lpotassium sodium tartrate tetrahydrate, 20 g/L NaOH) to all samples inthe PCR plates. The samples were then incubated at 95° C. for 10 min.After cooling down to room temperature, the absorbance at 405 nm wasmeasured. The residual activity (RA) was calculated using the followingformula:

RA (%)=(Abs(Stress))/(Abs(Ref))×100%

Abs(Stress): The absorbance at 405 nm of the sample in the stress MTP(incubated at 25, 26, 28 or 30° C.) after subtracting relevantbackground absorbance contributions.

Abs(Ref): The absorbance at 405 nm of the sample in the reference MTP(incubated at 4° C.).

Also, the half-lives for the degradation of each variant and parentendoglucanase were calculated using the following formula (by applying1st order kinetics for the degradation of EG):

${T\; 1\text{/}2} = {- \frac{{\ln (2)} \times T}{\ln \left( \frac{{Abs}({Stress})}{{Abs}({Ref})} \right)}}$

T: The incubation time.

Abs(Stress) and Abs(Ref): See above.

Half-life improvement factors (HIFs) can then be calculated as:

${HIF} = \frac{{T\; 1\text{/}2},{variant}}{{T\; 1\text{/}2},{wt}}$

T1/2, variant: The half-life for a specific variant

T1/2, wt (or wild-type): The half-life for EG wt (EG wild type), whereinsaid T1/2 wt is

T1/2 of the mature parent endoglucanase with SEQ ID NO: 2.

The HIFs of the tested purified variants are shown in Tables 2-5 below.HIFs in Tables 3-5 were calculated in the same way as in Table 2 exceptfor a slightly increased incubation temperature for variants andcontrols (26-30 degrees Celsius). All half-life values of the purifiedvariants were calculated relative to the half-life of GH9 wild-type(mature parent endoglucanase with SEQ ID No: 2) incubated at the samedetergent concentration and temperature measured as purified protein.HIF of GH9 wild-type in all tables is 1 (per definition).

TABLE 2 Purified variants of the mature parent GH9 endoglucanase (SEQ IDNO: 2) with corresponding half-life improvement factors (HIF) incubatedat 25° C. relative to a parent endoglucanase, e.g. an endoglucanase ofSEQ ID NO: 2. PUG Alteration as compared to (detergent Incubation SEQ IDNO: 2 concentration) time (hrs) HIF K507R 90% PUG 16 1.1 S17A 90% PUG 161.1 S63F 90% PUG 16 1.1 T694A 90% PUG 16 1.1 F20Y + A448S + T781M 90%PUG 16 1.3 Q834E 90% PUG 16 1.3 V56M 90% PUG 16 1.3 I602T 90% PUG 18 1.4A448E 90% PUG 16 1.4 A448W 90% PUG 16 1.5 F20G 90% PUG 16 1.5 I403Y 90%PUG 16 1.5 S538C 90% PUG 16 1.5 E408N 90% PUG 16 2.2 F20N 90% PUG 16 2.4K512P 90% PUG 16 2.5 E408G 90% PUG 16 2.9 P410G 90% PUG 16 2.9 E408A 90%PUG 16 3.0 F20P 90% PUG 16 3.3 E408P 90% PUG 16 4.2 E408S 90% PUG 16 5.4

TABLE 3 Purified variants of the mature parent GH9 endoglucanase (SEQ IDNO: 2) with corresponding half-life improvement factors (HIF) incubatedat 26° C. relative to a parent endoglucanase, e.g. an endoglucanase ofSEQ ID NO: 2. PUG Alteration as compared to (detergent Incubation SEQ IDNO: 2 concentration) time (hrs) HIF I302H 90% PUG 17.25 1.1 I387T 90%PUG 19.5 1.1 K51H 90% PUG 19.5 1.1 K754R 90% PUG 19.5 1.1 K390Q 90% PUG18 1.2 S605T 90% PUG 18.5 1.3 I602D 90% PUG 18.5 1.3 K51Q 90% PUG 18 1.6Y55M 90% PUG 18 1.6 K451S 90% PUG 18 1.9 Q416S 90% PUG 18 2.2 Q416D 90%PUG 18 2.5

TABLE 4 Purified variants of the mature parent GH9 endoglucanase (SEQ IDNO: 2) with corresponding half-life improvement factors (HIF) incubatedat 28° C. relative to a parent endoglucanase, e.g. an endoglucanase ofSEQ ID NO: 2. PUG Incubation Alteration as compared to (detergent timeSEQ ID NO: 2 concentration) (hrs) HIF K388R 90% PUG 5 1.3 T711Y 90% PUG5.5 1.8 T711V 90% PUG 5.5 1.9

TABLE 5 Purified variants of the mature parent GH9 endoglucanase (SEQ IDNO: 2) with corresponding half-life improvement factors (HIF) incubatedat 30° C. relative to a parent endoglucanase, e.g. an endoglucanase ofSEQ ID NO: 2. PUG Incubation Alteration as compared to (detergent timeSEQ ID NO: 2 concentration) (hrs) HIF E408D + K451S 90% PUG 16 26.1E408D + A448E 90% PUG 16 36.3 E408D 90% PUG 138 53.3 E408D + A448E 90%PUG 138 53.7 E408D + K451S 90% PUG 138 57.6 Y579W + E408D 90% PUG 13872.5

Example 4: Calculating Half-Lives and Half-Life Improvement Factors(HIF) for Xanthan Endoglucanase Variants

Half-life (T½ (in hours)) was calculated at a given detergentconcentration and storage temperature (Persil Universal Gel (PUG) 95%,30° C., 4 wk or more) for the wild-type controls and/or variants, as theresidual activity follows an exponential decay and the incubation time(hours) is known, i.e., according to the following formulas:

T1/2(variants)=(Ln(0.5)/Ln(RA-variants/100))*Time

T1/2(Wild-type)=(Ln (0.5)/Ln (RA-wild-type/100))*Time

A half-life improvement factor (HIF) is calculated as HIF=T½ (Variant)/T% (Wild-type), where the wild-type is incubated under the same storagecondition as the variant.

In the cases where the difference in stability between Wild-type andvariants is too big to accurately assess half-life for both Wild-typeand variant using the same incubation time, the incubation time forWild-type and variant is different e.g. 1 h for Wild-type and 672 h forthe most stable variants, Half-life Improvement Factor of variants werecalculated by HIF=T½ (Variant)/T % (Wild-type) where T½ of the WT was1.5 hr.

TABLE 6 Variant # Mutations HIF 1 S17A, F20P, N216D, A283D, H311N,E408D, 300 Y579W, I602T, A651P, A688G, T883R, F906A, Y934G, Q956Y 2F20P, I302D, S313D, E408D, D476R, Y579W, 310 S636K, T697G, V756Y, V881Q,T887K, F906A, A937E 3 F20P, S313D, E408D, Y579W, S636K, A688G, 375T697G, N905D, A937E 4 F20P, I302D, S313D, E408D, D476R, Y579W, 442S636K, T697G, W719R, V756Y, V881Q, T887K, F906A, A937E 5 N216Q, S313D,E408D, D476R, Y579W, I602T, 521 F638N, A651P, T697G, W719R, R880K,T887K, K921R, Y934G 6 N216D, S313D, E408D, D476R, A564E, Y579W, 553I602T, F638N, A651P, Y690F, T697G, W719R, V756H, N833D, A869V, R880K,V881T, T887K, K921R, S928D, Y934G, T999R 7 F20P, I302D, S313D, E408D,D476R, Y579W, 557 S636K, T697G, W719R, V756Y, N848D, A869V, V881Q,T887K, N905D, F906A, Q912V, A937E, T999R, F1048W 8 F20P, I302D, S313D,E408D, D476R, Q489P, 566 Y579W, S636N, T697G, W719R, V756Y, A824D,N848D, V881Q, T887K, F906A, S928D, A937E 9 F20P, I302D, S313D, E408D,Q416S, D476R, 597 Q489P, Y579W, I602T, S636K, A651P, T697G, W719R,V756Y, A824D, N833D, N848D, T883R, T887K, F906A, A937E 10 F20P, A186P,I302D, S313D, E408D, D476R, 616 Q489P, Y579W, A599S, I602T, S636K,A651P, T697G, W719R, V756Y, N848D, T883R, T887K, F906A, A937E 11 N216D,S313D, E408D, D476R, Y579W, I602T, 622 V603P, F638N, A651P, A688G,T697G, W719R, V756H, R880K, T887K, K921R, S928D, Y934G, K948R 12 F20P,K51Q, I302D, S313D, E408D, D476R, 640 Q489P, Y579W, I602T, S636K, A651P,T697G, W719R, V756Y, N848D, T883R, T887K, F906A, A937E 13 F20P, I302D,S313D, A346D, E408D, D476R, 644 Q489P, Y579W, S636N, T697G, W719R,V756Y, A824D, N848D, V881Q, T887K, F906A, A937E, T999R 14 F20P, I302D,S313D, E408D, D476R, Q489P, 655 Y579W, I602T, S636N, T697G, W719R,V756Y, A824D, N848D, V881Q, T887K, N905D, F906A, A937E, T999R, A1037E,F1048W 15 F20P, K51Q, I302D, S313D, E408D, D476R, 658 Q489P, Y579W,I602T, S636K, A651P, T697G, W719R, V756Y, A824D, N848D, T883R, T887K,F906A, S928D, A937E, A1037E 16 N216D, S313D, A346D, E408D, D476R, Q489P,663 A559P, Y579W, I602T, F638N, A651P, A688G, T697G, W719R, V756H,R880K, T887K, K921R, S928D, Y934G 17 F20P, I302D, S313D, A346D, E408D,D476R, 663 Q489P, Y579W, I602T, T697G, W719R, V756Y, N848D, V881Q,T887K, F906A, A937E 18 N216D, S313D, E408D, D476R, Q489P, A559P, 679Y579W, I602T, F638N, A651P, A688G, T697G, W719R, V756H, Q834E, R880K,T887K, T892P, K921R, S928D, Y934G 19 F20P, I302D, S313D, E408D, D476R,Q489P, 693 A559N, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y,N848D, T883R, T887K, F906A, S928D, A937E 20 F20P, I302D, S313D, E408D,D476R, Q489P, 702 Y579W, I602T, S636K, A651P, T697G, W719R, V756Y,N848D, T883R, T887K, F906A, A937E 21 F20P, I302D, S313D, E408D, Q416S,D476R, 720 Q489P, A559N, Y579W, I602T, S636K, A651P, T697G, W719R,V756Y, N848D, T883R, T887K, F906A, A937E 22 F20P, I302D, S313D, E408D,D476R, Y579W, 769 I602T, S636N, T697G, W719R, V756Y, A824D, N848D,V881Q, T887K, F906A, S928D, A937E, T999R, F1048W

1. A detergent composition comprising an endoglucanase variant, whereinthe variant comprises an alteration at one or more positions in a regionselected from the group consisting of: i) region 10 corresponding toamino acids 1 to 94 of SEQ ID NO: 2, ii) region 11 corresponding toamino acids 106 to 114 of SEQ ID NO: 2, iii) region 12 corresponding toamino acids 139 to 209 of SEQ ID NO: 2, iv) region 13 corresponding toamino acids 252 to 266 of SEQ ID NO: 2, v) region 14 corresponding toamino acids 302 to 338 of SEQ ID NO: 2, vi) region 15 corresponding toamino acids 362 to 546 of SEQ ID NO: 2, vii) region 16 corresponding toamino acids 596 to 611 of SEQ ID NO: 2, viii) region 17 corresponding toamino acids 661 to 805 of SEQ ID NO: 2, ix) region 18 corresponding toamino acids 829 to 838 of SEQ ID NO: 2, x) region 19 corresponding toamino acids 1043 to 1055 of SEQ ID NO: 2, and xi) combinations thereof;wherein said variant has at least 60% and less than 100% sequenceidentity to SEQ ID NO:
 2. 2. The detergent composition comprising anendoglucanase variant according to claim 1, wherein said variant has atleast 61%, at least 62%, at least 63%, at least 64%, at least 65%, atleast 66%, at least 67%, at least 68%, at least 69%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto SEQ ID NO:
 2. 3. The detergent composition comprising anendoglucanase variant according to claim 1, wherein said alteration atone or more positions is selected from the group consisting ofalterations in positions: 17, 20, 51, 53, 55, 56, 60, 63, 79, 87, 186,192, 302, 311, 313, 387, 388, 390, 403, 408, 410, 416, 448, 451, 471,472, 476, 489, 507, 512, 515, 538, 598, 599, 602, 605, 609, 676, 688,690, 694, 697, 698, 699, 711, 719, 754, 756, 760, 781, 786, 797, 833,834, 835, 1048, and combinations thereof of SEQ ID NO: 2, whereinnumbering is according to SEQ ID NO:
 2. 4. The detergent compositioncomprising an endoglucanase variant according to claim 1, wherein saidalteration at one or more positions is selected from the groupconsisting of: 517A, F20P, F20N, F20G, F20Y, K51Q, K51H, E53P, E53G,Y55M, V56M, Y60F, S63F, T87R, A186P, K192N, 1320D, 1302H, 1302V, 1302M,H311N, S313D, 1387T, K388R, K390Q, 1403Y, E408D, E408S , E408P, E408A,E408G, E408N, P410G, Q416S , Q416D, A448E, A448W, A448S, K451S, G471S,S472Y, D476R, Q489P, K507R, K512P, S515V, S538C, Y579W, S598Q, A599S,1602T, 1602D, V603P, 5605T, G609E, D676H, A688G, Y690F, T694A, T697G,R698W, T699A, T711V, T711Y, W719R, K754R, V756H, V756Y, 5760G, T781M,N786K, T797S, A824D, N833D, Q834E, S835D, F1048W, and combinationsthereof, wherein numbering is according to SEQ ID NO:
 2. 5. Thedetergent composition comprising an endoglucanase variant according toclaim 1, wherein said alteration at one or more positions is selectedfrom the group consisting of alterations in positions: 17, 20, 302, 311,313, 408, 476, 602, 688, 697, 719, and combinations thereof whereinnumbering is according to SEQ ID NO:
 2. 6. The detergent compositioncomprising an endoglucanase variant according to claim 1, wherein saidalteration at one or more positions is selected from the groupconsisting of alterations in positions: 517A, F20P, 1302D, H311N, S313D,E408D, D476R, 1602T, A688G, T697G, W719R, and combinations thereofwherein numbering is according to SEQ ID NO:
 2. 7. The detergentcomposition comprising an endoglucanase variant according to claim 1,wherein the variant further comprises an alteration at one or morepositions selected from the group consisting of alterations inpositions: 216, 283, 346, 559, 564, 579, 636, 638, 651, 824, 848, 869,880, 881, 883, 887, 892, 905, 906, 912, 921, 928, 934, 937, 948, 956,999, 1037, and combinations thereof wherein numbering is according toSEQ ID NO:
 2. 8. The detergent composition comprising an endoglucanasevariant according to claim 1, wherein said alteration at one or morepositions is selected from the group consisting of: N216D, N216Q, A283D,A346D, A559P, A559N, A564E, Y579W, S636K, S636N, F638N, A651P, A824D,N848D, A869V, R880K, V881T, V881Q, T883R, T887K, T892P, N905D, F906A,A912V, K921R, S928D, Y934G, A937E, K948R, Q956Y, T999R, A1037E, andcombinations thereof wherein numbering is according to SEQ ID NO:
 2. 9.The detergent composition comprising an endoglucanase variant accordingto claim 1, wherein the variant comprises one or more of the followingset of substitutions: F20Y + A448S + T781M E408D + K451S E408D + A448EE408D Y579W + E408D


10. The detergent composition comprising an endoglucanase variantaccording to claim 1, wherein the variant comprises one or more of thefollowing set of substitutions: S17A, F20P, N216D, A283D, H311N, E408D,Y579W, I602T, A651P, A688G, T883R, F906A, Y934G, Q956Y F20P, I302D,S313D, E408D, D476R, Y579W, S636K, T697G, V756Y, V881Q, T887K, F906A,A937E F20P, S313D, E408D, Y579W, S636K, A688G, T697G, N905D, A937E F20P,I302D, S313D, E408D, D476R, Y579W, S636K, T697G, W719R, V756Y, V881Q,T887K, F906A, A937E N216Q, S313D, E408D, D476R, Y579W, I602T, F638N,A651P, T697G, W719R, R880K, T887K, K921R, Y934G N216D, S313D, E408D,D476R, A564E, Y579W, I602T, F638N, A651P, Y690F, T697G, W719R, V756H,N833D, A869V, R880K, V881T, T887K, K921R, S928D, Y934G, T999R F20P,I302D, S313D, E408D, D476R, Y579W, S636K, T697G, W719R, V756Y, N848D,A869V, V881Q, T887K, N905D, F906A, Q912V, A937E, T999R, F1048W F20P,I302D, S313D, E408D, D476R, Q489P, Y579W, S636N, T697G, W719R, V756Y,A824D, N848D, V881Q, T887K, F906A, S928D, A937E F20P, I302D, S313D,E408D, Q416S, D476R, Q489P, Y579W, I602T, S636K, A651P, T697G, W719R,V756Y, A824D, N833D, N848D, T883R, T887K, F906A, A937E F20P, A186P,I302D, S313D, E408D, D476R, Q489P, Y579W, A599S, I602T, S636K, A651P,T697G, W719R, V756Y, N848D, T883R, T887K, F906A, A937E N216D, S313D,E408D, D476R, Y579W, I602T, V603P, F638N, A651P, A688G, T697G, W719R,V756H, R880K, T887K, K921R, S928D, Y934G, K948R F20P, K51Q, I302D,S313D, E408D, D476R, Q489P, Y579W, I602T, S636K, A651P, T697G, W719R,V756Y, N848D, T883R, T887K, F906A, A937E F20P, I302D, S313D, A346D,E408D, D476R, Q489P, Y579W, S636N, T697G, W719R, V756Y, A824D, N848D,V881Q, T887K, F906A, A937E, T999R F20P, I302D, S313D, E408D, D476R,Q489P, Y579W, I602T, S636N, T697G, W719R, V756Y, A824D, N848D, V881Q,T887K, N905D, F906A, A937E, T999R, A1037E, F1048W F20P, K51Q, I302D,S313D, E408D, D476R, Q489P, Y579W, I602T, S636K, A651P, T697G, W719R,V756Y, A824D, N848D, T883R, T887K, F906A, S928D, A937E, A1037E N216D,S313D, A346D, E408D, D476R, Q489P, A559P, Y579W, I602T, F638N, A651P,A688G, T697G, W719R, V756H, R880K, T887K, K921R, S928D, Y934G F20P,I302D, S313D, A346D, E408D, D476R, Q489P, Y579W, I602T, T697G, W719R,V756Y, N848D, V881Q, T887K, F906A, A937E N216D, S313D, E408D, D476R,Q489P, A559P, Y579W, I602T, F638N, A651P, A688G, T697G, W719R, V756H,Q834E, R880K, T887K, T892P, K921R, S928D, Y934G F20P, I302D, S313D,E408D, D476R, Q489P, A559N, Y579W, I602T, S636K, A651P, T697G, W719R,V756Y, N848D, T883R, T887K, F906A, S928D, A937E F20P, I302D, S313D,E408D, D476R, Q489P, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y,N848D, T883R, T887K, F906A, A937E F20P, I302D, S313D, E408D, Q416S,D476R, Q489P, A559N, Y579W, I602T, S636K, A651P, T697G, W719R, V756Y,N848D, T883R, T887K, F906A, A937E F20P, I302D, S313D, E408D, D476R,Y579W, I602T, S636N, T697G, W719R, V756Y, A824D, N848D, V881Q, T887K,F906A, S928D, A937E, T999R, F1048W


11. The detergent composition comprising an endoglucanase variantaccording to claim 1, wherein said endoglucanase variant has activity onxanthan gum pre-treated with xanthan lyase.
 12. The detergentcomposition comprising an endoglucanase variant according to claim 1,wherein said variant has an improved stability in a detergentcomposition compared to a wild-type endoglucanase having a sequenceidentical to that of SEQ ID NO:
 2. 13. The detergent compositioncomprising an endoglucanase variant according to claim 1, wherein saidvariant has a half-life improvement factor (HIF) of >1.0 relative to awild-type endoglucanase having a sequence identical to that of SEQ IDNO:
 2. 14. (canceled)
 15. A method of washing an article comprising:applying the detergent composition of claim 1 to an article; wherein thearticle is a laundry article, a surface, or combinations thereof; anddegrading xanthan gum from the article.